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Home , Anxiogenic, Bicuculline, Convulsant, Epipregnanolone, Gabazine, GABA receptor antagonist

Umeå University Medical Dissertations New Series No 1099- ISSN 0346 6612 ISBN 978-91-7264-273-7 From the department of Clinical Sciences, Obstetrics and Gynecology Umeå University

Effects of neuroactive on the recombinant GABAA in Xenopus oocyte

Mozibur Rahman

Umeå 2007 Department of Clinical Sciences, Obstetrics and Gynecology Umeå University SE-901 85 Umeå, Sweden

Cover illustration: The Revealed, created by Graham T. Johnson for the Howard Hughes Medical Institute Bulletin

Copyright© 2007 by Mozibur Rahman ISSN 0346-6612 ISBN 978-91-7264-273-7 Printed by Print & Media, Umeå University, 2007

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To my family and parents

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Contents

page Abstract 7 List of original papers 8 Abbreviation 9 Introduction 10 1. Relationship between the neuroactive metabolites of sex and stress hormones and disorders 10 2. of naturally occurring neuroactive steroids acting on GABAA receptors 12 2.1 Synthesis of 3α-OH steroids 13 2.2 Synthesis of 3β-OH steroids and 13 2.3 Synthesis of 5α-androstane 3α,17β-diol 14 2.4 Synthesis of THDOC 14 3. Concentration of 3α/3β neuroactive steroids in plasma and brain 16 4. GABAA receptor 16 4.1 GABAergic system 16 4.2 GABAA receptor subunit combinations and their proportion 17 4.3 Distribution of GABAA receptor in the brain 18 4.4 Pharmacological property of GABAA receptor 19 4.4.1 Allsosteric effect 19 4.4.2 Partial agonist and ‘superagonists’ of GABAA receptor 19 4.4.3 20 4.5. and GABAA receptor 20 2+ 4.6 Zn and GABAA receptor 21 4.7. Mechanism of GABAA receptor kinetics (deactivation desensitization, synaptic and extrasynaptic, phasic and tonic, duration of channel opening) 21 4.8 Pharmacological effects depend on GABA receptor subunit composition 23 4.9 GABAA receptor and disorders 24 5. Neuroactive agonists 25 5.1 Synaptic effect of agonist neuroactive steroids 25 5.2 Extrasynaptic effect of agonist neuroactive steroids 27 5.3 Subunit dependence of neuroactive steroids 28

4 5.4 Structure activity relationship 29 5.4.1 Backbone structure of neuroactive steroids 29 5.4.2 Steroid- Mimetic Core 30 5.4.3 Replacing C5-position 30 5.4.4 Replacing C11-position 30 5.4.5 Replacing C20-position 31 5.4.6 Replacing C21-position 31 5.4.7 Steroid nucleus 31 5.5 Binding sites of neuroactive steroids 31 5.6 Physiological role of neuroactive steroids 32 5.7 Neuroactive steroids as potential 33 5.7.1 effect of neuroactive steroids 33 5.7.2 Effect of neuroactive steroids in learning and memory 34 5.7.3 Effect of neuroactive steroids on sleep 34 5.7.4 Effect as 35 5.8.5 Analgesic effect of neuroactive steroids 35 6. Neuroactive steroid antagonists 35 6.1 and other sulfated steroids 35 6.1.1 Modulation of GABAA receptor by pregnenolone sulfate 35 6.1.2 Mechanism of action of pregnenolone sulfate 36 6.1.3 Site of action of pregnenolone sulfate 37 6.2 3β-hydroxy steroids 37 6.2.1 Mode of action of 3β-hydroxy steroids 37 6.2.2 Modification of 3β-position 38 6.2.3 Mechanism of action of 3β-hydroxy steroids 39 7. Recombinant GABA receptor: Advantages and disadvantages with studies on recombinant receptors expressed in oocyte over cell lines 40 8. Uses of rodent models for the testing of drugs for preclinical evaluation 41 Aims 42 Method 43 Chemicals 43 cDNA cloning 43 In vitro transcription and expression in the Xenopus oocyte 43 Oocyte electrophysiology 44 Data analysis 45 Statistical analysis 45 Results 46 GABA site antagonism and inhibition by is

5 not dependant on α-subunit (Paper I) 46 Pregnenolone sulfate response in not dependant on γ-subunit (Paper II) 46 Pregnenolone sulfate and Zn2+ do not share the same binding site (Paper II) 46 Functional differences between 3β-hydroxysteroids and pregnenolone sulfate (Paper III) 47 The neuroactive steroids activity is different between the recombinant human and rat α1β2γ2L GABAA receptors (Paper IV) 47 Neuroactive steroids activity differs between 2L (long form) and 2S (short from) variant of γ-subunit of human GABAA receptor (Paper IV) 48 Discussion 48 GABA and pentobarbital inhibition by bicuculline at α1-, α4-, 5- subunit containing GABAA receptor 48 Pregnenolone sulfate response is not dependant on γ-subunit 49 3β-hydroxysteroids and pregnenolone sulfate inhibit recombinant rat GABAA receptor through different mechanisms 50 The neuroactive steroids activity between the human and rat α1β2γ2L recombinant GABAA receptors 50 Differential response of neuroactive steroids activity between 2L (long form) and 2S (short from) variant of γ-subunit of human GABAA receptor 51 Conclusions 52 Acknowledgements 53 References 55 Appendix Paper I - IV

6 Abstract Introduction: Neuroactive steroids represent a class of both synthetic and naturally occurring steroids that have an effect on neural function. In addition to classical genomic mechanism by the hormones , deoxycorticosterone and , the 3α- OH metabolites of these hormones enhance GABAA receptor through rapid non-genomic mechanism. The site(s) of action of these neuroactive steroids namely 3α-OH-5α-pregnan- 20 one (3α5αP), (3α,5α)-3,21-deoxycorticosterone(3α5α-THDOC) and 5α androstane- 3α,17β-diol on GABAA receptor are distinct from that of and binding sites. The modulation site(s) has a well-defined structure activity relationship with a 3α-hydroxy and a 20-ketone configuration in the pregnane molecule required for agonistic action. Pregnenolone sulfate is a noncompetitive GABAA and inhibit GABA activated Cl- current in an activation dependant manner. 3β-hydroxy A-ring reduced pregnane steroids are also GABAA receptor antagonist and inhibit GABAA receptor function and its potentiation induced by their 3α-diesteromers in a noncompetitive manner. Aim: The aim was to investigate if the effect of GABA, pentobarbital antagonism by bicuculline and if the effect of GABA-agonist and antagonist neuroactive steroids including pregnenolone sulfate is dependant on the α-subunits of GABAA receptor. Furthermore, the studies aimed at investigating the binding site of pregnenolone sulfate and if its effect is dependent on γ-subunit. In addition, the inhibitory effect of pregnenolone sulfate and 3β- hydroxy steroids has been characterized. We also wanted to investigate if the neuroactive steroids effect vary between the human and rat recombinant α1β2γ2L receptors and between the long (L) and short (S) variants of γ2-subunit. Method: Experiments were performed by the two electrodes voltage-clamp technique using oocytes of Xenopus laevis expressed with recombinant GABAA receptors containing α1, α4 or α5, β2, γ2L and γ2S-subunits. Results: There was no difference between the α1, α4 and α5-containing subunits regarding GABA and pentobarbital inhibition by bicuculline. GABA-activated current in the binary αβ was potent than that of ternary αβγ receptor. Unlike Zn2+ effect, inhibition by pregnenolone sulfate on the GABAA receptor is not dependant on the γ-subunit. It is likely that the 2’ residue closest to the N-terminus of the protein at M2 helix on both α1 and β2 subunit are critical to the inhibitory actions of PS and the function of Cl- channels. Point mutation at M2 helix of the β2-subunit (β2A252S) can dramatically reduce the inhibitory effect of PS on the GABAA receptors without affecting the inhibitory properties of 3β- hydroxysteroids. Agonist and antagonist steroids also varied in their efficacy between the human and rat α1β2γ2L receptor. Neuroactive steroids also showed difference between human γ2L and γ2S-containing receptor. Conclusions: GABA and pentobarbital antagonism by bicuculline is not dependant on α- subunit. Pregnenolone sulfate binding site is different from that of Zn2+. 3β- hydroxysteroids and pregnenolone sulfate inhibit GABAA receptor through different mechanisms. Neuroactive steroids also differ between species and between the long and short variant of γ- subunit. Key words GABA, GABAA receptor, neuroactive steroids

7 Lists of original papers*

Paper I. GABA-site antagonism and pentobarbital actions do not depend on the α-subunit type in the recombinant rat GABA-A receptor. Rahman, M., Zhu, D., Lindblad, C., Johansson, I., Holmberg, E., Isaksson, M., Taube, M., Bäckström, T., Wang, M., 2006. Acta physiol 187, 479-488.

Paper II. Pregnenolone sulfate and inhibit recombinant rat GABAA receptor through different channel property. Wang, M., Rahman, M., Zhu, D., Bäckström, T., 2006. Acta physiol 188, 153 - 163.

Paper III. 3β-hydroxysteroids and pregnenolone sulfate inhibit recombinant rat GABAA receptor through different channel property. Ming-De Wang, Mozibur Rahman, Di Zhu, Inga-Maj Johansson and Torbjörn Bäckström. European journal of 557, 124 -131.

Paper IV. Functional difference between recombinant human and rat α1β2γ2L GABAA receptors. Mozibur Rahman, Monica Isaksson, Inga-Maj Johansson, Gianna Ragagnin, Torbjörn Bäckström and Ming-De Wang, 2007. Manuscript.

* All published papers are reproduced in this thesis with the kind permission of the copyright holders

8 Abbreviations GABA γ-amino DBI binding inhibitor 3α5αP 3α, 5α-tetrahydroprogesterone, 3α-OH-5α-pregnan-20-one, 3α5βP 3α-OH-5β-pregnan-20-one, 3α5αTHDOC 3α, 5α-tetrahydrodeoxy- 3α5βTHDOC 3α, 5β-tetrahydrodeoxy-corticosterone NMDA N-methyl-D-asparate PMDD premenstrual dystrophic disorder P450scc P-450 side chain cleavage 3α-HSD 3α-hydroxysteroid dehydrogenase 3β-HSD 3β-hydroxysteroid dehydrogenase 17β-HSD 17β-hydroxysteroid dehydrogenase 5α-R 5α-reductase 11β-HSD 11β-hydroxysteroid dehydrogenase 11βOHase 11β-hydroxylase 8-HSOR 18-hydroxysteroid oxidoreductase 21OHase 21-hydroxylase StAR steroidogenic acute-regulatory 3α-OH 3α-hydroxyl 3β-OH 3β-hydroxyl PS pregnenolone sulfate DHEAS sulfate 3β5αP/UC1010 3β-OH-5α -pregnan-20-one, Isoallopregnanolone 3β5βP/UC1014 3β-OH-5β -pregnan-20-one, UC1011 5α-pregnan-3β, 20β-diol UC1013 5β-pregnan-3β, 20α-diol UC1015 5β-pregnan-3β, 21-diol-20-one; 3β5βTHDOC UC1019 5α-pregnan-3β, 20α-diol UC2020 5β-pregnan-3β, 20β-diol 3α-adiol/3α5αADL 5α-androstane-3α,17β-diol DOC deoxycorticosterone 3α5αACN 3α-5α- androstane carbonitrile DMSO dimethyl sulfoxide ANOVA analysis of variance BMI bicuculline methiodide IPSC inhibitory post-synaptic current sIPSC spontaneous inhibitory post-synaptic current PKC protein kinase C DGC dentate gyrus granule cells CGC cerebellar granule cells

Imax the maximum current amplitude

EC50 concentration of that produces 50% of Imax

9

Introduction

1. Relationship between the neuroactive metabolites of sex and stress hormones and disorders The sex steroid hormones, and progesterone, influence a variety of behaviors in vertebrate and have a crucial effects on female reproductive function (Whitfield et al., 1999). Estrogen is required for the development of female phenotype, sexual maturation, female genital function and skeletal maintenance. Progesterone is necessary for conception and the maintenance of pregnancy. In adult women, the main sources of are the granulose cells of developing follicle and the corpus luteum in the ovary. Progesterone is synthesized mainly in the granulose cells of corpus luteum, also in the placenta and the adrenal gland (Speroff, 2005). Two progesterone receptors (PR) are also known, PRA and PRB (Alves et al., 1998; Bethea, 1993). These hormones are known to act through genomic mechanism in which intracellular receptor are located in the nucleus or cytoplasm and act as -activated transcription factors in the regulation of gene expression (Fig. 1). However, metabolites of progesterone and several stress hormones have effects through membrane bound receptors such as ion channels; a fast and non-genomic mechanism in which receptor binding to DNA and RNA synthesis is not required (Fig. 1) (Baulieu and Robel, 1995; Frye et al., 1992; Rupprecht, 2003). While the action of steroids at the genome requires a time period from minutes to hours that is limited by the rate of protein biosynthesis (McEwen, 1991), the modulatory effects of metabolites are fast occurring events requiring only milliseconds to seconds (McEwen, 1991). There is increasing evidence that these metabolites which act via non-genomic pathways may alter neuronal excitability (Lambert et al., 1995; Majewska et al., 1986; Paul and Purdy, 1992). The term ‘neuroactive steroids’ has been coined for these metabolites with this particular property (Paul and Purdy, 1992). The 3α-reduced metabolites of progesterone and deoxycorticosterone namely, 3α, 5α-tetrahydroprogesterone (3α5αP; allopregnanolone) and 3α, 5α-tetrahydrodeoxy-corticosterone (3α5αTHDOC) were the first steroids that have been shown to modulate neuronal excitability via their interaction with γ-aminobutyric acid type A (GABAA) receptors (Majewska et al., 1986). Several other like glutamate (N-methyl-D-asparate, NMDA) (Wu et al., 1991), nicotinic (Valera et al., 1992), muscarinic (Klangkalya and Chan, 1988), (Parry, 2001) and the system (Halbreich, 2003) are also modulated by such steroids (Fig. 1). Acting on the GABAA receptor the and metabolites may therefore exert their clinically significant effects in certain women; at low concentration, moderate to severe adverse emotional reactions are induced in up to 20% individual (Beauchamp et al., 2000; Fish et al., 2001), namely negative mood in

10 premenstrual dystrophic disorder (PMDD) (Backstrom et al., 2003; Sundstrom Poromaa et al., 2003) and petit mal (Banerjee and Snead, 1998; Grunewald et al., 1992), catamenial epilepsy -the epileptic in

Figure 1. Genomic and non-genomic site of action of steroid hormones and their metabolites.

women related to plasma estrogen and progesterone during the menstrual cycle (Backstrom, 1976). At higher doses they affect learning (Johansson et al., 2002), act as , anti-aggressive, /anesthetic and anti-epileptic effect in both animal and human (Backstrom et al., 1990; Bjorn et al., 2002; Paul and

11 Purdy, 1992; Wang et al., 2001). The main focus of this thesis is the effect of these steroid metabolites (neuroactive steroids) on the GABA-system.

2. Biosynthesis of naturally occurring neuroactive steroids acting on GABAA receptors Neuroactive steroids are formed de novo in and glia, or generated by metabolism of circulating precursors that originate in peripheral steroidogenic

Figure 2. The pathway for synthesis of allopregnanolone and pregnenolone sulfate within the or glial cell. Steroidogenic acute-regulatory (StAR) protein might interact with the mitochondrial receptor (MBR) to facilitate the transport of across the mitochondrial membrane. Abbreviations: 3α-HSD, 3α-hydroxysteroid dehydrogenase; 3β- HSD, 3β-hydroxysteroid dehydrogenase; 5α-DHP, 5α-dihydroprogesterone; P450scc, P450 side-chain cleavage. (Adapted from Belelli and Lambert, 2005).

12 organs (Compagnone and Mellon, 2000). The neuroactive steroids are A-ring reduced metabolites of the steroid hormones progesterone, deoxycorticosterone, and testosterone (Compagnone and Mellon, 2000). Their synthesis in the brain is controlled by the endogenous peptide-diazepam binding inhibitor (DBI), a ligand for the ‘peripheral’ benzodiazepine binding site, which are independent of GABA binding sites (Costa et al., 1994). Synthesis of different groups of neuroactive steroids is described in the next paragraph. Brain astrocytes and neuron express cytochrome P-450 side chain cleavage cytochrome (P450scc) which converts cholesterol to pregnenolone an immediately necessary for the synthesis of all hormonal steroids (Fig. 2) (Patte-Mensah et al., 2003; Zwain and Yen, 1999). Steroidogenic acute-regulatory protein (StAR) also facilitates the conversion. 3β-hydrxysteroid dehydrogenase (3β-HSD) converts pregnenolone to progesterone. Other that mediate the synthesis of neuroactive steroids are cytochrome P-450c17 (P450c17); 3α-hydroxysteroid dehydrogenase (3α-HSD); 17β hydroxysteroid dehydrogenase(17β-HSD); 5α-reductase(5α-R); 5α- (5α-DHT); 11β-hydroxysteroid dehydrogenase(11β-HSD); Sulfotransferase and sulfatase;11β-hydroxylase (11βOHase); 18-hydroxylase (18βOHase); 18-hydroxysteroid oxidoreductase (18-HSOR); 21- hydroxylase(21OHase) (Mensah-Nyagan et al., 1999).

2.1 Synthesis of 3α-OH steroids For the synthesis of allopregnanolone (3α-OH-5α-pregnan-20-one) from progesterone, 5α-reductase and 3α-hydroxysteriod dehydrogenase are needed, whereas its 5β-steroid-isomer, pregnanolone (3α-OH-5β-pregnan-20-one) is produced by the enzymatic activity of 5β-reductase and 3α-hydroxysteroid dehydrogenase (3α-HSD) (Fig. 2). Because the activity of the 3α-HSD is far greater than that of the 5α-reductase, steroid 5α-reductase is the rate-limiting steps in the biosynthesis of the neuroactive steroids (Penning et al., 1985; Russell and Wilson, 1994). Allopregnanolone and pregnenolone have high affinity to the GABAA receptor (Majewska et al., 1986). Allopregnanolone has been found to be the most potent of the progesterone metabolite, followed by pregnenolone (Paul and Purdy, 1992), (Zhu et al., 2001). Allopregnanolone persist in the brain after adrenalectomy and gonadectomy or after pharmacological suppression of adrenal and gonadal suppression (Cheney et al., 1995; Corpechot et al., 1993; Purdy et al., 1991), indicating that this steroid can be synthesized de novo in the brain via 5α- reduction of progesterone.

2.2 Synthesis of 3β-OH steroids and pregnenolone sulfate Pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS) are also endogenously produced GABA active steroids (Paul and Purdy, 1992).

13 Pregnenolone sulfate is synthesized from pregnenolone and mediated by the sulfotransferase (Fig. 2). Conversion from dehydroepiandrosterone to dehydroepiandrosterone sulfate (DHEAS) is also mediated by sulfotransferase. Dehydroepiantrosterone is metabolized from pregnenolone by cytochrome P450C17. Epipregnanolone (3β-OH-5β -pregnan-20-one) and other 3β-OH are also evident to be synthesized in the body (Hill et al., 2001). They are synthesized from O O O

5α−reductase 3β-HSD

O O HO H H β α Progesterone 5α-DHP 3 5 P (Isoallopregnanolone) Figure 3. Synthesis of 3β-OH steroids (Isopregnanolone) progesterone. 5α or 5β-dihdroprogesterone is synthesized from progesterone by enzyme 5α-or 5β-reductase. 3β-hydroxysteroid dehydrogenase (3β-HSD) is essential for the conversion to 3β-hydroxy steroids. Isoallopregnanolone (3β-OH- 5α -pregnan-20-one) is produced in the same way to allopregnanolone with the exception that 3β-HSD is needed in stead of 3α-HSD (Fig. 3).

2.3 Synthesis of 5α-androstane 3α, 17β-diol Synthesis of potent dihydrotestosterone from the precursor testosterone is mediated by steroid 5α-reductase. Breakdown to the inactive androgen metabolite 5α- androstane-3α,17β-diol (3α-adiol) is mediated by reductive 3α-HSD OH OH OH

5α-reductase 3α-HDS

RoDH1 O OH O H H Testosterone Dihydrotestosterone 5α-Androstane-3α,17β-diol Figure 4. Synthesis of 5α-androstane 3α,17β-diol. RoDH1 represents the rat retinol dehydrogenase 1 enzyme.

(Fig. 4) (Biswas and Russell, 1997). 5α-androstane-3α,17β-diol (3α-adiol) also modulate the GABA-mediated Cl- flux (Frye et al., 1996)

2.4 Synthesis of THDOC Stress hormone (3α,5α)-3,21-deoxycorticosterone(3α5α-THDOC) and (3α,5β)- 3,21-deoxycorticosterone(3α5βTHDOC) are also potent naturally occurring steroids which acts on the GABAA receptor (Crawley et al., 1986; Gasior et al., 1999; Lambert et al., 2001; Majewska et al., 1986). 3α5αTHDOC is a metabolite of

14 the deoxycorticosterone and is responsible for the sedative and anti-seizure activity of deoxycorticosterone in animal models (Reddy and Rogawski, 2002). Deoxycorticosterone (DOC) can also be metabolized from progesterone and this conversion is mediated by steroid 21 hydroxylase (P45021)(Edwards et al., 2005). The neuroactive steroid 3α5α- tetrahydrodeoxycorticosterone (3α5αTHDOC) is synthesized from deoxycorticosterone by two sequential A-ring reductions. 5α-Reductase first converts deoxycorticosterone to the intermediate 5α-dihydrodeoxycorticosterone, OH OH OH

O O O O α− 5 3α-HSD P45021 reductase O O O HO H H Progesterone Deoxycorticosterone 5α-DHDOC 3α5αTHDOC Figure 5. Synthesis of 3α5αTHDOC.

which is then further reduced by 3α-hydroxysteroid oxidoreductase to form 3α5αTHDOC (Fig. 5). 3α5αTHDOC has significant sedative effects acting on GABAA receptors. Plasma concentrations of 3α5αTHDOC (half-life <20 min) increase rapidly following systemic administration of deoxycorticosterone. In contrast to the progesterone-derived neuroactive steroid allopregnanolone, which

O O CN O O

HO HO HO H H HO H 3α5αACN alphaxalone Δ4 pregnen- 3α ol-20 one UC1010

OH HO O HO HO

H H H

HO HO HO HO H H H H UC 1011 UC1015 UC1019 UC1020

Figure 6. Other agonist and antagonist steroids tested in this project (chemical names are given in the method part)

15 is synthesized de novo in the brain, 3α5αTHDOC appears to be derived nearly exclusively from the adrenal cortex. The conversion of deoxycorticosterone into 3α5αTHDOC occurs both in peripheral tissues and in the brain (Reddy, 2003).

3. Concentration of 3α/3β neuroactive steroids in human plasma and brain (mean ± SEM) Steroids Plasma(nM) Brain(nM) Follicular Luteal Postmeno Luteal -pausal progesterone 5.0 ± 0.50a 34.7 ± 2.40 65d 137d 3α-OH-5β-pregnan-20-one 0.6 ± 0.00b 1.1 ± 0.50 - 114d 3α-OH-5α-pregnan-20-one 0.53 ±0.19c 2.14 ± 0.37 47d 66d 3β-OH-5α-pregnan-20-one 0.29 ± 0.14c 1.23 ± 0.28 - - 3β-OH-5β-pregnan-20-one 0.09 ± 0.08c 0.26 ± 0.13 - - pregnenolone sulfate 11.2 ± 0.6 a 15.2 ± 0.8 a - 100f pregnenolone *2.19g - - - 3α5α-androstane-3α,17β-diol *0.475g - - - Data are cited from following references: a (Wang et al., 1996); b(Sundstrom et al., 1998); c (Havlikova et al., 2006); d(Bixo et al., 1997); e (Bixo et al., 1995); f(Lanthier and Patwardhan, 1986) ; g(Kancheva et al., 2007).* concentration in adult men.

To our knowledge there is no report on the plasma concentration of 3α5αTHDOC in human. Although Hill et al. (2007) measured the plasma level of 3α5αTHDOC (0.3 nM/L) in adult men (unpublished, personal contact). However, in rat the plasma level normally fluctuate between 1 and 5 nM but increase to 15- 40 nM following acute stress (Reddy and Rogawski, 2002) and can reach 40-60 nM in pregnancy (Concas et al., 1998). Purdy et al. first observed that both circulating and brain level of 3α5αTHDOC reach their peak during stress; in the rat this amounts to a circulating concentration of ~20 nM and a brain tissue concentration of ~120 nM (40 ng/g) (Purdy et al., 1991). The plasma level of level in human is around 0.8- 1.2 µM/L. In stressful situation like PhD examination the cortisol level increased up to ~1.3 - 1.8 µM/L (Droogleever Fortuyn et al., 2004).

4. GABAA receptor 4.1 GABAergic system As mentioned earlier that GABA mediates most inhibitory transmission events in the vertebrate brain. GABA is synthesized by two isoforms of decarboxylase (GAD) (Erlander et al., 1991; Esclapez et al., 1994; Soghomonian and Martin, 1998). Inhibition mediated by GABA is involved crucially in both

16 short- and long-term regulation of neuronal excitability. Three different types, GABAA, GABAB and GABAC, of GABA receptor might be activated in the CNS (Sivilotti 1993). It has been estimated that approximately 33% of the in the mammalian are GABAergic (Purvez et al., 2004). GABAA receptors are members of the ligand gated ion channel superfamily (Schofield et + 2+ al., 1987). Metabotropic GABAB receptor is coupled to certain K and Ca channels via GTP binding protein (G-protein) and/or other messengers (Hill and Bowery, 1981). GABAA receptor is bicuculline sensitive and GABAB is beclofen sensitive. The third type of GABA receptor, insensitive to both bicuculline and beclofen was designated GABAC (Drew et al., 1984). The GABAC responses are also of the fast type associated with the opening of an anion channel; they are, unaffected by typical modulators of GABAA receptor like benzodiazepines and barbiturates (Bormann and Feigenspan, 1995; Sivilotti and Nistri, 1991).

4.2 GABAA receptor subunit combinations and their proportion To date, 16 isoforms of GABAA receptor have been identified (Whiting, 2003). These comprise α1-6, β1-3, γ1-3, δ, ε, π and θ. GABAA receptors are pentameric proteins (Nayeem et al., 1994) of five different subunits containing 2α/2β/1γ- or δ-subunit variants(Fig. 7) (Farrar et al., 1999; Klausberger et al., 2001; Knight et al., 2000). Immunological, pharmacological, and functional analysis give the evidence that the α1β2γ2 combination represents the largest population of GABAA receptor (~60%) followed by α2β3γ2 (~15-20%) and α3βnγ2 (~10-15%,

Figure 7. GABAA receptor with binding sites of neuroactive steroids, barbiturate, benzodiazepine and . Right figure shows the transmembrane domains of five subunits. Note that the second transmembrane domain of each subunit forms the channel lining.

17 n=1,2 or 3) (Fig. 8) (Fritschy and Brunig, 2003; Mohler et al., 2004; Mohler et al., 2002; Wallner et al., 2003). Receptors containing the α4-, α5-, and α6-suunits, as well as the β1-,γ1-γ3, δ-,π- and θ-subunits, form minor receptor population. α4βnδ, α4βnγ and α6β2,3γ2 receptors- each of these is less than 5% of all GABAA receptors (Fig. 8). α6βnδ has a small population only in the cerebellum. Likewise, α6β2,3γ2 receptors are located only in the cerebellum. The ρ-subunits are expressed primarily in the retina and correspond to the so-called GABAC receptor (Bormann, 2000). Structurally, this receptor differs from classical GABAA receptors in that the channel comprises a homopentamer of ρ-subunit rather than heteropentamers.

4.3 Distribution of GABAA receptor in the brain Expression of GABAA receptor subtypes in the adult brain exhibits a remarkable region- and neuron-specificity which suggests that individual subtypes are present in distinct neuronal circuits. α1β2γ2 receptor present in most brain areas and it is localized to interneuron in the and cortex (layer I-IV), and cerebral Purkinje cells (McKernan and Whiting, 1996). α2β3γ2 receptor present in cerebral cortex (layer I-IV), hippocampal formation, amygdale, striatum, olfactory bulb, hypothalamus, superior colliculi and motor nuclei (Fritschy and Brunig, 2003). α3βnγ2, α3γ2, α3θ are abundant in the cerebral cortex (layers V-VI), amygdala, olfactory bulb, thalamic reticular and intralaminar nuclei, superior colliculus, brainstem, spinal cord, locus coeruleus etc. α4βxδ in the dentate gyrus and thalamus. α5β3γ2 receptor is localized in hippocampal pyramidal cells, deep

Figure 8. Proportion of GABAA receptor in the central nervous system (CNS) (based on the report by Mohler et al., 2004 and Fritschy et al., 2003)

18 cortical layers, amygdala, olfactory bulb, hypothalamus, superior colliculus, superior olivary nucleus, spinal trigeminal nucleus and spinal cord. α6β2,3γ2, α6β2,3δ and α6β2,3γ2 are found mainly in the cerebellum, dorsal cochlear nucleus (Fritschy and Brunig, 2003). Other minor combinations are distributed throughout the brain.

4.4 Pharmacological property of GABAA receptor 4.4.1 Allsosteric effect

The GABAA receptor can be modulated by a number of therapeutic agents, including benzodiazepines (Macdonald and Olsen, 1994; Sieghart, 1992), barbiturates (Smith and Riskin, 1991), anaesthetics, (Harris et al., 1995), Zinc (Smart, 1992) and neuroactive steroids (Hawkinson et al., 1994; Puia et al., 1990). Pharmacological analysis of GABAA receptors revealed that α-subunits govern GABA affinity (Smith et al., 2001), α and γ subunits regulate benzodiazepine site pharmacology (Buhr et al., 1996; McKernan et al., 1995; Smith et al., 2001; Wingrove et al., 1997). In fact, the binding pocket of benzodiazepines is located between the α and γ subunit (Sigel, 2002). The α1-, α2-, α3-, and α5- GABAA receptors are diazepam-sensitive receptors, whereas the α4- and α6- GABAA receptors are insensitive to diazepam (Benson et al., 1998; Wingrove et al., 2002). α1-, α2-, α3-, and α5 receptors are distinguished further by their affinity to (α1 > α2 = α3 >> γ5) and various β-carbolines (α1 > α2= α3) (Mohler et al., 1996; Sieghart, 1995). The action of anticonvulsant loreclazole and intravenous anesthetics are also potentiators of GABAA receptors (Hill- Venning et al., 1997; Wingrove et al., 1994). Neuroactive steroids are also positive allosteric modulators of recombinant and native GABAA receptors (Lambert et al., 2001) and will be described in detail in the next section. Furthermore, novel ligands like 3-Heteroaryl-2-pyridones and ROD compounds are being introduced, which exhibit intrinsic activity only on specific GABAA receptor subtypes (Collins et al., 2002; Sigel et al., 2001). Neurochemical, electrophysiological, and behavioral evidence accumulated over the past two decades suggests that GABAA receptors also mediate certain acute and chronic actions of ethanol (Deitrich et al., 1989; Harris et al., 1998; Ueno et al., 2001). Similar to the GABAA receptors modulators mentioned above, ethanol exhibits an array of central actions, including anxiolytic, anticonvulsant, sedative-, , and general anesthetic effects, in a dose-dependent manner (Deitrich et al., 1989; Frye et al., 1981). However, some recent attempts have failed to reproduce all these findings (Borghese et al., 2006; Carta et al., 2004).

4.4.2 Partial agonist and ‘superagonists’ of GABAA receptor and THIP (4,5,6,7-tetrahydroisoxazole(5,4-c) pyridine-2-ol) are widely used as selective GABAA receptor agonist (Chebib and Johnston, 2000). THIP

19 (commonly named ), a conformationally restricted analogue of muscimol, is a potent partial agonist of high efficacy (Krogsgaard-Larsen et al., 1977). It has no effect on synaptic currents at concentration up to 3 µM in α1- containing receptor; a full agonist at α2-containig receptor whereas in the extrasynaptic receptors it is potent, high efficacious ‘super-agonist’ (Ebert et al., 2002; Ebert et al., 1994; Wafford and Ebert, 2006). Another GABA agonist 4- PIOL(5-(4-piperidyl)isoxazol-3-ol), is a low efficacy partial agonist (Ebert et al., 2002). On the other hand, Compound 5b (N, N’-1, 4-butanediylbis[4- aminobutanamide]) also produce a maximum response that was 150% that of GABA and was described as ‘super-agonist’ (Carlier et al., 2002).

4.4.3 Channel blocker Bicuculline, Zn2+, TBPS, picrotoxin and pregnenolone sulfate are the antagonists of GABAA receptor. Bicuculline is the competitive blocker of GABA. In other word it is GABA site antagonist (Krishek et al., 1996; Polenzani et al., 1991). Picrotoxin, Zn2+ and pregnenolone sulfate etc exert their effect directly binding in the ion channel (Eisenman et al., 2003; Hosie et al., 2003; Krishek et al., 1996). Pregnenolone sulfate is considered as the open Cl- channel blocker, whereas Zn2+ stabilizes the closed Cl- channel and has no effect on open Cl- channel. Picrotoxin causes a decrease in mean channel open time. It works by preferentially shifting opening channels to the briefest open state (1 msec). Experimental like and the cage convulsant t-butyl bicyclophosphorothionate (TBPS) also act to block the Cl- channel (Delorey and Olsen 1994 -Basic Neurochemistry, Chapter 18).

4.5. Barbiturates and GABAA receptor Electrophysiological and neurochemical studies have shown that general anesthetic agents can have three mechanism of action, namely (i) a potentiation of the GABA response (Evans, 1979; Study and Barker, 1981), (ii) a direct activation of GABAA receptor (Franks and Lieb, 1994; Robertson, 1989) and (iii) at high concentrations, a block of the GABA chloride channel (Robertson, 1989; Schwartz et al., 1986). The affinities and efficacies of potentiating and direct effect of pentobarbital vary with GABAA receptor subtypes. Potentiating effect over GABA EC20 is higher in the α6-subunit containing receptor compare to α1- subunit containing receptor (Thompson et al., 1996). α6-subunit also shows higher affinity and efficacy for direct effect of pentobarbital. Like α-subunits, β-subunits are also known to contribute to the effects of barbiturates on GABA receptor (Belelli et al., 1999; Thompson et al., 1996; Wafford et al., 1996). In general, the β2- and β3- subunits confer greater sensitivity and efficacy to pentobarbital than the β1-subunit for both its allosteric and agonist actions. Direct effect of pentobarbital was blocked by picrotoxin but not by competitive antagonist

20 bicuculline or SR95531, indicating that direct agonist activity of pentobarbitone is not mediated via the GABA binding site (Thompson et al., 1996). Many studies have been performed for searching the site of action of barbiturates. A number of residues within the first and second transmembrane domains (TMs) of the β-subunit have been identified as sensitive to positive modulation and/or direct activation by several anesthetics, including the barbiturates (Birnir et al., 1997; Carlson et al., 2000; Cestari et al., 2000; Chang et al., 2003; Dalziel et al., 1999; Pistis et al., 1999; Serafini et al., 2000). Another report showed that a single amino acid residue located in the N-terminal domain of the α-subunit, regulates pentobarbital efficacy (Drafts and Fisher, 2006).

2+ 4.6 Zn and GABAA receptor The transition metal ion Zn2+ is concentrated into zinc-containing neurons in the CNS and is thought to be involved in modulating the sensitivity of GABA and glutamate receptors to their respective neurotransmitters (Frederickson, 1989; Frederickson and Bush, 2001). In some neurons, the sensitivity of GABAA receptors to Zn2+ varies throughout development (Brooks-Kayal et al., 2001; Smart, 1992; Taketo and Yoshioka, 2000), and Zn2+ could be critically involved in temporal-lobe epilepsy, in which GABAA receptor subunit expression may be altered (Buhl et al., 1996; Gibbs et al., 1997; Molnar and Nadler, 2001). Zn2+ ions inhibit GABAA receptor function by an allosteric mechanism that is critically dependent on the receptor subunit composition: αβ-subunit combinations show the highest sensitivity, and αβγ-isoforms are the least sensitive, whereas αβδ subunits and αβε receptor showing intermediate sensitivities to Zn2+(Krishek et al., 1998; Saxena and Macdonald, 1996). Zn2+ antagonism is also affected by α- subunit (Draguhn et al., 1990; Smart et al., 1991; White and Gurley, 1995). Inhibitory effect of Zn2+ is reduced by exchanging α1-subunit for α2- or α3- subunits. Recently, the site of action of Zn2+ has also been elucidated. Three discrete sites that mediate Zn2+ inhibition have been identified. One is located within the ion channel, and the other two are on the external amino (N)-terminal face of the receptor at the interfaces between α- and β-subunits (Hosie et al., 2+ 2003). Low Zn sensitivity of GABAA receptors containing the γ2-subunit results from disruption to two of the three sites after receptor subunit co- assembly.

4.7. Mechanism of GABAA receptor kinetics (deactivation desensitization, synaptic and extra-synaptic, phasic and tonic, duration of channel opening GABAA receptor, like other receptors in the brain may be synaptic or extrasynaptic depending on the location of the receptors. The receptors within the synaptic cleft (bouton) are known as ‘synaptic’ whereas the receptors that are over the neuronal surface outside the synaptic cleft are known as the ‘extra-synaptic’

21 receptors. Receptors containing a γ2-subunit in association with α1-, α2- or α3- subunits (α1β2/3γ2, α2β2/3γ2 and α3β2/3γ2) are the predominant synaptic receptor subtypes (Farrant and Nusser, 2005). Receptors that contain α4-, α5- or α6- subunits (α6βnδ, α4βnδ and α5βnγ2) are predominantly or exclusively extrasynaptic (Fritschy and Brunig, 2003; Nusser et al., 1998). In synapse, each vesicle is thought to liberate several thousand GABA molecules into the synaptic cleft, produce a response (IPSC) at high concentration of GABA (0.3-1.0 mM) and they are short lived (10-100 ms) (Mozrzymas et al., 2003; Semyanov et al., 2004). There are only few number of receptors (from ten to a few hundred) clustered opposite the release site (Brickley et al., 1999; Mody et al., 1994; Nusser et al., 1997). Synaptically released GABA acting on postsynaptic GABAA receptors produces “phasic” inhibition, whereas “tonic” inhibition results from the continuous activation of extrasynaptic receptors by ambient GABA (Farrant and Nusser, 2005). The main feature of phasic GABAA receptor-mediated inhibition is the rapid synchronous opening of a relatively small number of channels that are clustered at the synaptic junction, whereas tonic inhibition results from random, temporally dispersed activation of receptors that are distributed (albeit in a potentially non-uniform manner) over the neuronal surface. GABA-mediated tonic conductance are found in granule cells of the dentate gyrus (Farrant and Nusser, 2005; Nusser and Mody, 2002), thalamocortical relay neurons of the ventral basal complex (Porcello et al., 2003), layer V pyramidal neurons in the somatosensory cortex (Yamada et al., 2004), CA1 pyramidal cells (Bai et al., 2001), and certain inhibitory interneurons in the CA1 region of the hippocampus (Semyanov et al., 2003). Unlike receptors mediating phasic current, tonically active GABAA receptors show unusual high GABA affinity (Saxena and Macdonald, 1996) and be activated by the low ambient GABA concentrations (nanomolar to few micromolar) (Lerma et al., 1986; Tossman et al., 1986). At submicromolar concentrations, several competitive and non-competitive GABAA antagonists (, picrotoxin and bicuculline) reduced phasic currents, but had no effect on tonic currents. However, the antagonists blocked both phasic and tonic currents at high concentrations (Bai et al., 2001; Farrant and Nusser, 2005; Semyanov et al., 2003; Stell and Mody, 2002; Yeung et al., 2003). There is also difference in the tonic and phasic inhibitions by neuroactive steroids which will be discussed later. GABA receptor-mediated inhibitory postsynaptic currents (IPSCs) peak rapidly (0.5-5 ms) and usually decay with two time constants (slow and rapid decay) of ranging from few millisecond to tens or hundreds of milliseconds (Jones and Westbrook, 1995; Mozrzymas et al., 2003). A commonly observed use-dependent characteristic of GABAA receptors is desensitization. Desensitization can be defined macroscopically; as the 'decline in response in the continued presence of agonist'. Microscopically, it is essentially a 'long-lived agonist-bound closed state

22 of the receptor'. As ‘desensitized’ states are part of the generally agreed reaction mechanism for the GABAA receptor, exposure of the receptor to GABA will drive entry to these states; the time spent in these states will reflect the waveform of GABA exposure. With voltage-clamp of oocytes expressing GABAA receptor, the ability to apply agonist quickly is severely limited by the physical size of the oocyte, and so desensitization happens during the slow onset phase. This means that information relevant to the rapid onset of the IPSC is lost. Although such experimental stimuli are very different from the natural stimulus experienced by GABAA receptors in the brain, several physiologically relevant aspects of inhibition can be derived from this fundamental receptor property. As mentioned earlier the time course of the GABA concentration is transient and the decay of the IPSC is dominated by the ion channel closure that follows ligand removal, a macroscopic phenomenon known as deactivation.

4.8 Pharmacological effects depend on GABA receptor subunit composition There are evidences that several subunits are related to several behavioral effects. Transgenic mouse models enable exciting new perspectives for the development of pharmacology of modulating agents of GABAA receptor and their effect on different subunits. Benzodiazepines have two effects in the GABA system- sedative and anxiolytic. Histidine residue in α1, α2, α3, and α5 subunits is crucial for benzodiazepines binding. Sedative effect of benzodiazepines is mediated via the α1 subunit containing GABAA receptors (Rudolph et al., 1999). Replacement of histidine by arginine at position 101 at α1 subunit leads to mice in which the anxiolytic and muscle relaxant effects of diazepam are retained, whereas its effects on the motor component of sedation, its amnesic and anticonvulsant effects are abolished (McKernan et al., 2000; Rudolph et al., 1999). In contrast, the α2 subunit appears to be a major determinant for the anxiolytic and muscle relaxant effects of benzodiazepines (Crestani et al., 2001; Low et al., 2000). Agonistic activity at the α3 subunit also mediate the anxiolytic activity of benzodiazepines, although it occurred only at high receptor occupancy (Dias et al., 2005). Recently, it has been shown that the α5 subunit is important for sedative tolerance development to benzodiazepines and for acquisition and expression of associative memory and spatial learning (Collinson et al., 2002; Crestani et al., 2002; van Rijnsoever et al., 2004; Yee et al., 2004). In addition, α4 subunit is also implicated in the regulation of (Gulinello et al., 2001). A concentration-dependent decrease of the α4 subunit is seen after 4-day application of allopregnanolone to developing neuronal cells (Grobin and Morrow, 2000), whereas in the hippocampus and cerebellum, an increase in this subunit can be detected after withdrawal from chronic progesterone (or allopregnanolone) exposure and after short-term treatment (Concas et al., 1999; Follesa et al., 2001; Gulinello et al., 2001; Smith et al., 1998). α6 subunit is highly sensitive to pentobarbital effect

23 (Thompson et al., 1996) and neuroactive steroid (Belelli et al., 2002). In recent years, there has been an accumulating amount of evidence for the involvement of GABAA receptors in the action of anesthetics. The intravenous anesthetic etomidate shows GABAA subtype selectivity in vitro for β2- and β3-containing receptors when studied on recombinant receptors (Hill-Venning et al., 1997). A recent study shows that β3-containing receptors are the primary mediators of the anesthetic effects whereas β2, probably in combination with α1, and γ subunits mediates the sedating effects of etomidate (Wafford et al., 2004). The γ-subunit is thought to confer sensitivity of the receptor to benzodiazepines (Pritchett et al., 1989; Ymer et al., 1990). The γ1- and γ3-containing receptors have a 10-30 fold lower affinity for than do receptors containing γ2-subunit (Hadingham et al., 1995; Luddens et al., 1990; Wafford et al., 1993; Ymer et al., 1990). On the other hand, absence of γ subunit is essential for Zinc sensitivity (Hosie et al., 2003). The γ2-subunit is also involved in anxiety regulation, and it is changed during hormone manipulation and pregnancy (Concas et al., 1999; Crestani et al., 1999; Essrich et al., 1998; Follesa et al., 1998; Kittler et al., 2000). The γ2-subunit is essential for clustering of GABAA receptors and gephyrin and synaptic localization (Baer et al., 1999; Essrich et al., 1998) (Fritschy and Brunig, 2003; Schweizer et al., 2003). The δ-subunit is responsible for tonic conductance and important for neuroactive steroid modulation on GABAA receptor (Stell et al., 2003). Receptor knockout studies have revealed that the absence of the δ-subunit decreases the sensitivity to neuroactive steroids such as pregnanolone and alphaxalone, thereby influencing the duration of anesthesia and the anxiolytic effect of those steroids (Mihalek et al., 1999). The ε-subunit reduces neuroactive steroid and anesthetic modulation (Belelli et al., 2002; Davies et al., 1997; Thompson et al., 2002). Functionally, distinct subunit-specific properties have been identified in both recombinant and native receptors, supporting the concept that GABAA receptor heterogeneity is a major facet determining the functional properties of GABAergic inhibitory circuits (Mohler et al., 2001; Sieghart, 2000). In particular, the type of α- subunit determines the kinetics of receptor deactivation (Devor et al., 2001; Hutcheon et al., 2000; Verdoorn et al., 1990), and the presence of the δ-subunit results in markedly increased agonist affinity and apparent lack of desensitization (Adkins et al., 2001; Burgard et al., 1996; Fisher and Macdonald, 1997).

4.9 GABAA receptor and disorders When the balance between excitatory and inhibitory activity is shifted pharmacologically towards GABAergic transmission anxiolysis, sedation, amnesia, ataxia, and loss of consciousness can be induced. On the other hand, an attenuation of the GABAergic system results in arousal, anxiety, restlessness, , exaggerated reactivity, and even epileptic seizures. These GABAergic

24 system is known to multiple neurological and psychiatric diseases, including anxiety disorders (Malizia, 1999), epilepsy (Backstrom et al., 2003; Coulter, 2001; Duncan, 1999; Olsen et al., 1999), ethanol dependence (Morrow et al., 2001), Huntinton’s disease (Kunig et al., 2000), Angelman syndrome (DeLorey et al., 1998), and schizophrenia (Blum and Mann, 2002; Lewis, 2000; Nutt and Malizia, 2001). There is strong evidence that neuroactive steroid allopregnanolone and 3α5αTHDOC are involved in the pathophysiology of premenstrual syndrome, catamenial epilepsy, major depression, and stress-sensitive brain disorders. Neuroactive steroids PS and DHEAS have been shown to modulate memory functions. However, the significance of the testosterone-derived neuroactive steroid 3α-androstanediol is not well understood (Reddy, 2003).

5. Neuroactive steroid agonists Recent studies have indicates the existence of at least three effects of neuroactive steroids on GABAA receptor: one for the allosteric enhancement of GABA- evoked current, one for direct activation and one for the antagonistic action by the sulfated and 3β-OH steroids.

5.1 Synaptic effect of agonist neuroactive steroids As stated earlier GABA mediated inhibition may be of two varieties; synaptic and extrasynaptic. The brief inhibitory response of steroids that results from the activation of postsynaptic GABAA receptor is phasic response. Although both modes of operation clearly impact upon neuronal information processing at the cellular and network level, the extent to which each receptor pool influences brain excitability in normal and diseased states is not known. Synaptic GABAA receptors are ternary complexes that almost invariably incorporate the γ2-subunit in combination with α (mainly α1-, α2- and α3-) and β2/3-subunit isoforms. However, these receptor isoforms can also be located extra-synaptically (Farrant and Nusser, 2005). The kinetic of agonist steroids at synaptic GABAA receptor has been studied thoroughly by measuring the spontaneous inhibitory postsynaptic currents (sIPSC) from neurons in brain slices. The neurosteoids have little effect on the rise time or peak amplitude of the sIPSC, but they prolong the decay of IPSC (Haage et al., 2005). However this effect is neuron specific. In hippocampal CA1 neurons, cerebellar granule cells and Purkinje neurons, neuroactive steroids prolong the sIPSC at relatively low concentration (in nanomolar range) (Cooper et al., 1999; Harney et al., 2003; Vicini et al., 2002). On the other hand, micromolar concentrations are required to produce equivalent responses in hypothalamic neuron (Brussaard et al., 1997; Koksma et al., 2003). Alphaxalone enhance GABAA receptor modestly in hippocampus and not at all in the caudate nucleus. It enhances GABA binding in the temporal lobe better than the parietal and frontal lobe with no effect in the occipital lobe (Nguyen et al., 1995).

25 3α5αTHDOC enhanced GABA binding better in CA3 and subiculam than CA1 and entorhinal cortex (Nguyen et al., 1995).

Figure 9. Neuroactive steroids’ effect on the synaptic and extrasynaptic GABAA receptors and type of inhibition produced by them (adapted from Belelli and Lambert, 2005).

The effect of neuroactive steroids also depend on concentration (Haage and Johansson, 1999). In the dissociated neurons from the medial preoptic nucleus, 2µM 3α5αP modulating 100 µM GABA response is markedly depressed and the desensitization is faster, but the decay after GABA application is prolonged. In contrast, modulating 1 µM GABA response is markedly potentiated, the activation is faster, a prominent desensitization is induced, and the decay after GABA application is prolonged. Neurons in the same brain region can also show the heterogeneity. In hippocampal brain slices from 20-day-old rats, an approximately 30-fold higher concentration of pregnanolone (3α5βP) is required to prolong mIPSCs in dentate granule cells (DGCs) than in CA1 (Belelli and Lambert, 2005; Harney et al., 2003). Neuroactive steroid sensitivity to GABAA receptor of different brain regions also depend on the different stages of development. In

26 DGCs, mIPSCs that are recorded from 10-day-old rats are more sensitive than those from 20-day-old animals (Cooper et al., 1999). Recorded from HEK293 cells transfected with synaptic receptors and recombinant synaptic GABAA receptors showed that 3α5αP, 3α5αTHDOC and ACN ((3α5α17β)-3-hydroxyandrostane-17-carbonitrile) and B285 (3α5α17β)-3- hydroxy-18-norandrostane-17-carbonitrile) are the positive modulators of GABA site (Akk et al., 2004; Belelli et al., 2002; Reddy, 2003). These potentiating steroids prolong GABAergic inhibitory postsynaptic currents, although the amplitude is generally not increased (Haage et al., 2005). 3α5αP and probably other potentiating steroids are assumed to reduce the rate of GABA unbinding from the receptor (Haage et al., 2005).

At higher concentration (>10 µM), which occur during parturition (Stoffel- Wagner, 2003), neuroactive steroids directly activate the receptor (Majewska et al., 1986) similar to those of barbiturates, steroids but interact with different sites on GABAA receptor (Kerr and Ong, 1992). This ‘GABA-mimetic’ effect of neuroactive steroids is sufficient to suppress the excitatory neurotransmission (Shu et al., 2004).

5.2 Extrasynaptic effect of agonist neuroactive steroids The response of neuroactive steroids at relatively low concentration is mediated by the activation of extrasynaptic or perisynaptic α4, α5, α6 and δ-subunit containing GABAA receptors (Fig 9). These extra-synaptic receptors identified in hippocampal dentate gyrus granule cells (DGCs), the granule cells of the cerebellum and the relay neurons of the thalamus are distinct from their synaptic counterparts. Extrasynaptic conductance can have a considerable influence on neuronal excitability (Leroy et al., 2004). Such receptor assemblies exhibit both a high affinity for GABA and a reduced receptor desensitization in the continued presence of the agonist, properties that render these receptors ideally suited to sense the low ambient concentrations (~0.5-1 μM) of the estimated to be present extra-synaptically (Kennedy et al., 2002). These receptors are highly sensitive to neuroactive steroids in certain brain region. Low ‘physiological’ concentrations (10-100 nM) of 3α,5α-THDOC in mouse DGCs and CGCs, selectively enhance the tonic conductance, with little or no effect on the phasic conductance (Belelli and Lambert, 2005; Farrant and Nusser, 2005; Stell et al., 2003). GABAA receptor δ-subunit in a ternary combination (αβδ) contains α6- and α4-subunits (Farrant and Nusser, 2005; Fritschy and Brunig, 2003; Stell et al., 2003). Recombinant receptors containing the δ-subunit are highly sensitive to neuroactive steroid modulation (Wohlfarth et al., 2002). Tonic inhibition is reduced in δ subunit ‘knock-out’ mice, and the residual tonic current was insensitive to 3α5α-THDOC (Mihalek et al., 1999; Stell et al., 2003). The inhibitory

27 effect of neuroactive steroids differs in different brain region. 250 nM 3α5αTHDOC has no effect on the tonic inhibition in ventrobasalis complex of the thalamus (Porcello et al., 2003). Similarly, the tonic conductance of hippocampal CA1 neurons, which is mediated partly by receptors that contain the α5-subunit, is affected by 3α5αTHDOC above 100 nM (Belelli and Lambert, 2005; Farrant and Nusser, 2005; Stell et al., 2003). The modulation of tonic GABAA- receptor-mediated currents by neuroactive steroids is influenced by local neuroactive steroid metabolism, inhibition of which can greatly enhance the response of the tonic current in dentate granule cells to endogenous neuroactive steroids, while having no effect on the response to the synthetic metabolically stable neuroactive steroid ganaxalone (Belelli and Herd, 2003). Such neuroactive steroid metabolism demonstrates regional specificity, which could further contribute to the regional specificity of the tonic GABAA-receptor-mediated currents, and their modulation. This raises the intriguing possibility of selectively regulating network excitability with inhibitors of neuroactive steroid metabolism; these could thus have an antiepileptic action. In summary, evidence is emerging that the GABA mediated tonic conductance present in some neurons may have a considerable influence on neuronal signaling and network activity (Brickley et al., 2001; Hamann et al., 2002; Mitchell and Silver, 2003). The high sensitivity of neuroactive steroids in extrasynaptic receptor may represent an important target for these steroids.

Using labeled α- (Bgt) derivatives, it was observed in hippocampal neurons that the principal sites of both GABAA receptor endo- and exocytosis are extrasynaptic (Bogdanov et al., 2006). In addition, the synaptic GABAA receptors are recruited directly from their extrasynaptic counterparts, and constitute a dynamic mechanism for neurons to rapidly modulate receptor number at inhibitory synapses by controlling the availability and stability of extrasynaptic receptors.

5.3 Subunit dependence of neuroactive steroids As mentioned earlier that the GABA-ergic synaptic transmission may be differentially regulated by pregnane steroids in different brain regions, an effect that might be attributable to variations in GABAA receptor subunit composition. The α-subunit did not greatly influence the GABA-modulatory actions of 3α5αP, when co-expressed with β1 and γ2L subunits in Xenopus oocytes (Belelli et al., 2006). Likewise, the isoform of the β-subunit (1-3) has little influence on the GABA-modulatory actions of the pregnane steroids (Belelli et al., 2002; Hadingham et al., 1993; Sanna et al., 1997). The presence of γ-subunit is not a prerequisite for neuroactive steroid activity. In fact, the efficacy of 3α5αP modulation mediated by binary α1β1 is higher than that mediated by ternary

28 α1β1γ2 receptors. That is, the steroid to be more effective at the binary isoform, increasing the GABA-evoked response above the apparent maximal response to GABA (Belelli et al., 2002; Maitra and Reynolds, 1999). The isoform of the γ- subunit has little, or no, effect on the maximal GABA-modulatory effect of 3α5αP but significantly influences the potency of the steroid with “physiological concentrations” (3-30 nM). On the other hand, δ-subunit when coexpressed with α4- and β3- subunits, a receptor thought to be naturally present in the thalamus (Sur et al., 1999) shows high steroid sensitivity compare to γ- subunit containing receptor (Belelli et al., 2002; Davies et al., 1997). Receptors incorporating the ε- subunit are reported to be insensitive to the modulatory actions of the pregnane steroids, not the direct GABA-mimetic effect.

5.4 Structure activity relationship Structure-activity relationship of the neuroactive steroids and the effects associated with their use has been summarized in a number of articles (Hamilton 2002; Gyermek et al., 1968; Gyermek and Soyka, 1975; Laubach et al., 1955). The systemic investigation of several isomer combination of GABAA-active neuroactive steroids revealed, as a general rule that several features are of importance for an effective drug: geometry between ring A/B is crucial, Hydrogen bond donator in position 3, hydrogen-bond acceptor in position 20 and/or flexible bond at position 17 (Ragagnin et al., 2007, Purdy et al., 1990; Zorumski et al., 2000). By convention, the α- and β-refer to substituents below and above the plane of the steroid rings, respectively. In the next paragraphs, we describe the variation in the core and substitution in certain positions on the steroid nucleus. Modification of C3 position is described in the neuroactive steroid antagonists section.

5.4.1 Backbone structure of neuroactive steroids Anesthetic steroids typically have a saturated backbone of four rings, although this is not an absolute requirement for activity. These rings form a rigid framework for positioning the hydrogen bonding groups in three-dimensional spaces. Certain benz[e]indines have steroid like effect on neuron (Hamilton, 2002; Rodgers- Neame et al., 1992), even though they are tricyclic steroid analogues in which the steroid-A ring is opened and partially removed. The presence of hydrogen bond donor in the α-configuration at C3 and β-configuration at C17 are critical for anesthetic actions (Hawkinson et al., 1994; Hogenkamp et al., 1997; Purdy et al., 1990). These groups are important for the binding of steroids to a variety of proteins by means of hydrogen-binding with polar or charged residues (Brzozowski et al., 1997; Grishkovskaya et al., 2000). Replacing the hydrogen of hydroxyl with methyl, thus eliminating the ability of the steroid to donate a

29 hydrogen bond in this region, results in much reduction in potency (Upasani et al., 1997).

5.4.2 Steroid- Mimetic Core Replacing the steroid skeleton with an alicyclic framework gave a compound that showed weak potentiating activity (Burden et al., 1998). When the nitrile is replaced with an acetyl group the derivative became inactive (Hamilton, 2002).

5.4.3 Replacing C5-position The configuration at C5 is also important. Even if steroids with either 5α or 5β conformations are active, spatial difference in this position may affect the pharmacology of the neuroactive steroids. Studies with 5α-THDOC and its stereoisomer 5β-THDOC revealed important differences in potency, efficacy and regional selectivity at the GABAA receptor complex (Gee and Lan, 1991; Mennerick et al., 2004). Moreover, 5α reduced steroid, but not 5β-steroids, show a high degree of enantioselectivity/enantiospecificy in their action as modulator of GABAA receptor and as anesthetics (Covey et al., 2000). Although model has been proposed to account for steroids binding at the same site by invoking conformational changes (Hamilton, 2002; Purdy et al., 1990), it is also possible that they occupy different sites favored by their respective stereochemistries. Removal of angular methyl group of 5α -steroids i.e. 19-nor derivatives has variable effect on the potentiating activity of 5α and 5β-steroids, whereas incorporation of a methyl group at C5 eliminate or diminishes activity (Han et al., 1996).

5.4.4 Replacing C11-position The very potent but opposing effects of picomolar concentrations of cortisol and on GABA receptors in the intestine, with cortisol enhancing and cortisone reducing GABA responses, is very interesting both from a structure- activity and a physiological viewpoint. Cortisol and cortisone differ in structure only by the level of oxidation at carbon 11, cortisol being the 11β-hydroxy compound and cortisone the 11-oxo compound. 11-ketones like alphaxalone and 11α-amines i.e. minaxalone retain good activity and this is one of the few cases where polar groups are tolerated on the α-face of the steroid without serious loss potentiating activity (Hamilton, 2002). However in chloride uptake studies in cortical synaptosomes the 3α5α metabolite of cortisol and cortisone are antagonistic but enhance the positive allosteric effect of allopregnanolone (Stromberg et al., 2005).

30 5.4.5 Replacing C20-position Pregnan-20-one is easily metabolized to pregnan-20-ols. Reduction to 20α-or 20β- ol reduces the affinity and efficacy of pregnan steroids. However, a ketone is preferred 20-substituent for pregnanes for good its efficacy (Belelli et al., 1996; Hamilton, 2002; McCauley et al., 1995).

5.4.6 Replacing C21-position A variety of substituents can be accommodated at the C21-position, i.e. halides (Hill-Venning et al., 1996), (Xue et al., 1997) and thiosulfate (Fick et al., 1999) and heterocycles (Yang et al., 1999), generally without serious loss of activity. 3α5αTHDOC is a potent and efficacious GABAA , whereas the 21-OH analogues of pregnenolone are not. It rather antagonizes the effect of 3α5αP (Hamilton, 2002 ; Xue et al., 1997).

5.4.7 Steroid nucleus Andros-5-enes and pregn-5-enes with polar functional groups at the 17β- and C20 positions respectively retain good inhibitory activity (Hamilton, 2002).

5.5 Binding sites of neuroactive steroids Determining how neuroactive steroids interact with the GABAA receptor is a prerequisite for understanding their physiological and pathophysiological roles in the brain. Neuroactive steroids bind to GABAA receptors at a site that is distinct from the recognition sites for GABA, benzodiazepines, and barbiturates. This results in allosteric modulation of GABA binding or channel gating. Electrophysiological studies have confirmed that neuroactive steroids enhance chloride current through increasing both channel frequency and channel open duration (Callachan et al., 1987; Puia et al., 1990; Zhu and Vicini, 1997). Neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation (Akk et al., 2005). In a recent study, Hosie et al. (2006) identified two discrete binding sites in the receptor's transmembrane domains that mediate the potentiating and direct activation effects of neuroactive steroids. Their potentiating effect is mediated by a cavity formed by the α-subunit transmembrane domains. On the other hand, their direct activation is mediated by interfacial residues between α and β-subunits and is enhanced by steroid binding to the potentiation site. These profiles indicate that two distinct neuroactive steroid binding sites may exist; αTHr236 and βTyr284 residues in the transmembrane domain initiate direct activation whereas αGln241 and αAsn407 mediate the potentiating response (Hosie et al., 2006).

31 5.6 Physiological role of neuroactive steroids Many of the physiological actions of neuroactive steroids at the GABAA receptor complex have been evaluated (Majewska, 1987). As the neuroactive steroids are converted from gonadal hormone, it is often difficult to establish if the effect is due to parents hormonal steroids or their metabolites (Rupprecht et al., 1996). However, virtually all enzymes mediating these transformations have been identified in the CNS (Mellon et al., 2001). The concentrations of circulating neuroactive steroids vary during menstrual cycle, pregnancy (Bixo et al., 1997; Herbison, 2001), delivery and stress (Purdy et al., 1991). Therefore there is an obvious potential links with mood disorders such as stress induced depression and cognitive disturbance, PMDD and post-natal depression. In addition to modulating receptor function, there is evidence that treatment with steroids induce receptor plasticity through changes in expression of particular GABAA receptor subunits (Follesa et al., 2001; Gulinello et al., 2001). Following prolong administration pregnenolone causes expression of neuropeptide Y receptor Y1 gene in the amygdala, a region in the brain that is important for controlling anxiety (Ferrara et al., 2001). This is similar to the effect seen with ligands that bind to the benzodiazepine site of GABAA receptor and supports the hypothesis that there is a functional interaction between GABA and neuropeptide Y in the amygdala. Moreover, the endogenous benzodiazepine octadecaneuropeptide regulates corticotrophin-releasing hormone (CRH) mRNA expression, and this regulation is mediated by the effects of the peptide at GABAA receptors and positively influenced by the neuroactive steroids (Givalois et al., 1998). There is also evidence that allopregnanolone, at nanomolar concentration in vitro suppress the release of hypothalamic gonadotropine releasing hormone (Calogero et al., 1998). Other physiological actions mediated by the neuroactive steroids include the prevention of protein kinase C-dependant GABAA receptor in oxytocine neurons at the juvenile stage or during late pregnancy; after parturition GABAA receptors in oxytocine neurons are less sensitive to allopregnanolone (Brussaard et al., 2000); and possible effects on neurotransmission due to the catalepsy observed in mice following administration of allopregnanolone (Khisti et al., 1998). Side effects typical of benzodiazepines, such as tolerance, dependence and interactions with alcohol are also observed in prolong treatment of neuroactive steroids (Morrow et al., 2001). However, synthetic analogues appear to offer some advantages over the endogenous steroids with respect to their potential for abuse (Rowlett et al., 1999). Neuroactive steroids effect has also been reported in (NMDA) and sigma receptors (Baulieu, 1998; Mellon et al., 2001; Reddy and Kulkarni, 2000; Zorumski et al., 2000). Other actions are liable to be non-GABAergic include the suppression of pituitary follicle stimulating hormone from rat pituitary cells by 3α-hydroxy-4-pregnen-20-one and the blockage of voltage activated calcium channels in rat hippocampal and dorsal root

32 ganglion neurons by 3α-hydroxy-5α-androstane-17β-carbonitrile (Nakashima et al., 1998).

5.7 Neuroactive steroids as potential drugs A number of review articles have covered the potential that neuroactive steroids modulating GABAA receptor system might offer for treating various disorders (Majewska, 1992; Reddy and Kulkarni, 2000; Rupprecht et al., 2001; Zorumski et al., 2000). However, certain obstacles prevent the clinical use of endogenously occurring neuroactive steroids. Importantly, natural neuroactive steroids such as allopregnanolone have low bioavailability because they are rapidly inactivated and eliminated by glucoronide or sulfate conjugation at the 3α-hydroxy group. In addition, the 3α-hydroxy group of allopregnanolone may undergo oxidation to the ketone, restoring activity at receptors (Rupprecht et al., 1993). Ganaxalone (3α-hydroxy-3β-methyl-5α-pregnane-20-one), the 3β-methyl analogue of allopregnanolone, is an example of a synthetic neuroactive steroid congener that overcomes these limitation (Carter et al., 1997). Like allopregnanolone, ganaxalone is a positive of GABAA receptor. The following section will therefore focus on the therapeutic potential of the neuroactive steroids.

5.7.1 Anxiolytics effect of neuroactive steroids Neuroactive steroids modulate relatively to anxiety and stress. It has been proposed that their anxiolytic activity is a consequence of regulation of steroid biosynthesis via the hormones adrenocorticotropin hormone (ACTH) and corticotropine releasing hormones (CRH) (Torres et al., 2001). After an acute stress stimulus there is also a release of progesterone, pregnenolone, allopregnanolone and 3α5β-THDOC (Barbaccia et al., 1998; Purdy et al., 1992; Serra et al., 2000). Allopregnanolone and 3α5αTHDOC have been demonstrated to possess potent anxiolytic activity in several different animal anxiety models (Bitran et al., 1995; Crawley et al., 1986; Reddy and Kulkarni, 2000; Wieland et al., 1995). The anxiolytic effect has however never been shown in humans (Wihlback et al., 2006). A number of recent reports have actually indicated that allopregnanolone can induce aggression and anxiety (Fish et al., 2001; Gulinello et al., 2001; Miczek et al., 2003). A recent clinical study showed that the allopregnanolone and cortisol level are increased during the examination of PhD students (Droogleever Fortuyn et al., 2004). Therefore it has been suggested that allopregnanolone in certain individuals has biphasic effects; with low doses increasing an adverse, anxiogenic effect, and high doses decrease this effect and show beneficial, calming property. A major concern with potential new anxiolytics is whether they suffer the same drawbacks as classical benzodiazepine. Using the elevated plus-maze paradigm for assessing the anxiolytic activity, behaviorally

33 selective effects of neuroactive steroids have been reported which differ from those of the benzodiazepine diazepam (Rodgers and Johnson, 1998). Evaluation of novel neuroactive steroids as anxiolytics continues. Synthetic derivative Co 2- 6749 30, retains a 3β-trifluromethyl group that should block metabolism and enhance oral bioavailability, was selected for clinical development because there is a large separation between anxiolytic-like effect and side effects (Gasior et al., 1999; Vanover et al., 2000).

5.7.2 Effect of neuroactive steroids in learning and memory Hippocampus is a key brain area for learning and memory functions (Farr et al., 2000). Women with premenstrual dysphoric disorder often show difficulties in concentrating and develop fatigue during the luteal phase of the menstrual cycle; this is associated with high circulating levels of allopregnanolone (Sundstro and Backstrom, 1999). The enzymes needed for the production of allopregnanolone are also present in certain areas of the brain, notably the hippocampus (Compagnone and Mellon, 2000). Using the Morris water maze paradigm, allopregnanolone was found to inhibit learning (Johansson et al., 2002). 3β-steroid namely, 3β-20β-dihydroxy-5α-pregnane, antagonism at the GABAA receptor reduces the negative allopregnanolone effect on learning in the water maze (Turkmen et al., 2004). Pregnenolone sulfate infused into the basal magnocellualar nucleus enhance memory performance, whereas allopregnanolone disrupted memory (Mayo et al., 1993). Pregnenolone, DHEA and DHEAS increased memory when injected systemically, centrally or into the amygdala (Flood et al., 1992; Flood et al., 1988; Wolkowitz et al., 1995). There is evidence that the concentration of DHEA and DHEAS are decreased in patients suffering from Alzheimer’s disease (Hillen et al., 2000; Nasman et al., 1991; Sunderland et al., 1989). Thus neuroactive steroids should be further explored in the context of prevention of and/or treatment of age-related memory disorders.

5.7.3 Effect of neuroactive steroids on sleep As early as 1942, Selye reported the sedative and anesthetic properties of progesterone and some of its metabolites (Selye, 1942). 3α5αP, 3α5βP, 3α5αTHDOC exhibit benzodiazepine like effect, including reduced sleep latency and increased non-REM sleep with only small changes in slow wave and REM sleep (Bowers and Wehner, 1992; Olney et al., 1991). The limited bioavailability of steroids following oral administration is a significant issue in the development of these compounds as drug. However, several new compounds have been evaluated in animal models as potential and ; side effects for these activities are liable to be less severe than for anesthetics due to the reduced doses and a wider therapeutic margin. Tolerance to minaxadole and allopregnanolone has been reported at sedative doses following chronic treatment (Marshall et al.,

34 1997; Turkmen et al., 2006). Other compounds that have been evaluated as sedative hypnotics include Co 134444, Co 177843 and 177834 and Co 127501(Hamilton, 2002; Vanover et al., 1999; Vanover et al., 2001), CCD-3693 31 (Edgar et al., 1997), CCD-3693 is undergoing clinical evaluation for insomnia (Gasior et al., 1999).

5.7.4 Effect as anticonvulsant Neuroactive steroids are potent broad-spectrum anti-seizure agents. Several neuroactive steroids protect against seizure induced in animal GABAA receptor antagonist, picrotoxin induced seizures, and kindled seizures (Reddy, 2003; Reddy et al., 2004; Turner et al., 1989; Wieland et al., 1995). Some neuroactive steroids are highly effective in suppressing -,ethanol-, diazepam-, and neuroactive steroid-withdrawal seizures (Devaud et al., 1996; Devaud et al., 1995; Reddy et al., 2001). At very high doses, the neuroactive steroids also partially protect mice against maximal electroshock-induced seizures. Similarly, synthetic allopregnanolone analogues demonstrate comparable anticonvulsant efficacy (Carter et al., 1997; Hogenkamp et al., 1997). Ganaxalone has activity in a broad range of animal models of epilepsy. It has been shown to be well tolerated in adults and children (Nohria and Giller, 2007). The clinical studies with ganaxalone suggest the dose limiting side effect is sedation (Beyenburg et al., 2001). Withdrawal (Reddy and Kulkarni, 2000; Reilly et al., 2000), tolerance (Czlonkowska et al., 2001) and sedation (Kokate et al., 1994) are the associated with and neuroactive steroids that have been observed in rodent seizure model.

5.7.5 Analgesic effect of neuroactive steroids Recent reports indicate alphadalone and water soluble-amino steroids and their metabolite to be analgesic (Hamilton, 2002). However, related compound alphaxalone, which differs only in lacking a 21-OH group, is ineffective (Goodchild et al., 2001; Nadeson and Goodchild, 2001). The analgesic activity has been attributed to GABAA receptors in the spinal cord since the antinociception is reversed by administration of the GABAA antagonist bicuculline (Hamilton, 2002).

6. Neuroactive steroid antagonists 6.1 Pregnenolone sulfate other sulfated steroids 6.1.1 Modulation of GABAA receptor by pregnenolone sulfate Sulfated steroids like pregnenolone sulfate (PS), and dehydroepiandrosterone sulfate (DHEAS) can produce profound effects on behavior. PS, an abundant neuroactive steroid, enhances learning (Flood et al., 1995; Mayo et al., 1993) while antagonizing the impairment of learning and memory produced by ethanol and

35 (Melchior and Ritzmann, 1996). PS may play a role in cognition and have been reported as negative modulators of GABA receptors based on electrophysiological studies and GABA mediated 36Cl- uptake by rat brain synaptosomes (Demirgoren et al., 1991; Majewska et al., 1990). Although structural similarity of pregnenolone with that of 3α5α-steroids might lead one to expect that PS would share a common binding site with 3α5αP, this is not the case. Structure activity relationships for GABAA receptor modulation are different for sulfated inhibitory steroids . nonsulfated potentiating steroids (Park-Chung et al., 1999). Potentiation by nonsulfated steroids requires 3α stereochemistry. Pregnenolone is inactive at GABAA receptors. In contrast, PS is inhibitory, both as the 3α and 3β isomers. Although the addition of a negatively charged sulfate or hemisuccinate group at the C3 position converts a number of neuroactive steroids from potentiating to inhibitory (Park-Chung et al., 1999), a negatively charged group at C3 is not absolutely essential for inhibition, as the nonsulfated neuroactive steroid dehydroepiandrosterone (DHEA) is also inhibitory, although less potent than its sulfated derivative DHEAS (Imamura and Prasad, 1998; Park- Chung et al., 1999). Some steroids, such as 11-keto derivative of PS is of particular interest, as it can behave as a positive, negative, or neutral modulator. It was suggested that 11-ketopregnenolone sulfate (11-ketoPS) exerts a dual action on distinct positive and negative steroid modulatory sites associated with the GABA receptor (Park-Chung et al., 1999).

6.1.2 Mechanism of action of pregnenolone sulfate The mechanism of inhibition by pregnenolone sulfate has been studied extensively. Inhibition of GABAA receptor function by PS and DHEAS is not voltage-dependent (Gibbs et al., 2006; Majewska et al., 1988; Spivak, 1994), indicating that these sulfated steroids do not need to penetrate significantly into the membrane field to reach their sites of action, and suggesting that they act allosterically rather than by physically occluding the ion pore of the GABAA receptor. Pregnenolone sulfate has been reported to act by reducing channel- opening frequency (Mienville and Vicini, 1989). A subsequent single-channel patch clamp study of α1β2γ2L GABAA receptors expressed in HEK293 cells found that PS had no effect on the rate constants for channel closing and opening, and concluded PS produces a block that is independent of GABA binding. Channel activation state, or membrane potential, with an IC50 of 100 nM, suggesting that PS acts essentially as a slow noncompetitive antagonist (Akk et al., 2001). In contrast, studies of macroscopic GABAA receptor currents have reported that PS modulates synaptic transmission by enhancing GABA receptor desensitization (Haage et al., 2005; Shen et al., 2000). The disagreement between microscopic and macroscopic studies of PS inhibition of GABAA receptor function remains to be resolved (Gibbs et al., 2006).

36

6.1.3 Site of action of pregnenolone sulfate The site of action of sulfated neuroactive steroids on GABAA receptors remains unclear. Based upon the observation that PS reduced the apparent affinity of 35S- TBPS, Sousa and Ticku (1997) suggested that PS and DHEAS might bind at the picrotoxin/cage convulsant site. However, a mutation to the transmembrane M2 channel domain eliminated picrotoxin sensitivity but the inhibitory effects of PS and DHEAS persisted (Gibbs et al., 2006; Shen et al., 1999). The absence of voltage sensitivity or alteration of single-channel open time argues against a binding site within the pore. Akk et al. (2001) identified a valine residue in the channel domain of the α1 subunit that slowed the development of PS inhibition when mutated to , but concluded that this residue is unlikely to be part of the binding site and likely influences PS action indirectly (Akk et al., 2001). A recent study in C. elegans identified multiple residues in transmembrane domain 1 (M1), as well as a residue near the extracellular end of the M2 helix, that are critical for low-μM inhibition of C. elegans GABAA receptors by PS. This latter residue is of particular interest, as it is a positively charged arginine that could potentially coordinate with the negatively charged sulfate of PS. The C. elegans receptor exhibits some pharmacological differences as compared to mammalian GABAA receptors (for example, pregnanolone is inhibitory), so these results may or may not be relevant to mammalian receptors; however, it is notable that an arginine residue is also found in this region of mammalian GABAA receptor subunits (Gibbs et al., 2006; Wardell et al., 2006).

Block of responses to high-efficacy agonists by this sulfated steroid is greater than block of responses to partial agonists at saturating concentrations (Eisenman et al., 2003; Wang et al., 2002). This is called “activation dependant” or “state dependant inhibition”. Picrotoxinin, another GABA channel blocker superficially similar to pregnenolone sulfate in its activation dependence but the site of action of pregnenolone sulfate does not require a functional picrotoxin site for inhibition of GABA responses (Shen et al., 1999). GABA receptor antagonist Zn2+ also acts in activation dependant manner. However, Zn2+ binding site is located in the interface between α- and β-subunits and is activity is minimal in ternary αβγ receptor (Hosie et al., 2003).

6.2 3β-hydroxy steroids 6.2.1 Mode of action of 3β-hydroxy steroids Considerable effort has been given to characterize the effect of 3β- hydroxysteroids. For potentiating activity, OH-group at 3α-stereochemistry is essential regardless of the configuration at C5. An advantage of incorporating 3β- substituents is that potential hormonal activity resulting from oxidative

37 metabolism is abolished, and oral bioavailability is improved (Hogenkamp et al., 1997). Several earlier studies explored 3β-steroids as potential steroid antagonists. [3H]Flunitrazepam binding assays suggest that 3β-hydroxysteroids competitively antagonize the actions of the potentiating steroids (3α,5α)-3-hydroxypregnan-20- one (3α5αP) and (3α,5β)-3-hydroxypregnan-20-one (3α5βP), two standard representatives of the 5α-reduced and 5β-reduced classes of potentiating steroids (Prince and Simmonds, 1992, 1993). In electrophysiological studies, 3β5βP antagonized the 3α5βP-induced enhancement of GABA current (Maione et al., 1992; Maitra and Reynolds, 1998). However, only 3β5αP diminished the inhibitory effects of 3α5αP and 3α5βP on population spikes evoked in rat hippocampal CA1 stratum pyramidal (Wang et al., 2000). Studies on the chloride uptake into synaptosomes in rat cerebral cortex, in hippocampus and sIPSP in medial preoptic nucleus (MPN) showed that 3β-steroids reduced the 3α5αP-enhanced GABA response (Stromberg et al., 2006). In the presence of only GABA and absence of 3α5αP, some of the 3β-steroids potentiated GABA-evoked chloride ion uptake and prolonged the spices decay time, whereas the others had little or no effect on GABA stimulated current (Stromberg et al., 2006).

6.2.2 Modification of 3β-position Along with α in C5, 3β-ethyl, -propyl, -trifluoromethyl and -(benzyloxy)methyl, as well as substituents of the form 3β-XCH2, where X is Cl, Br, or I or contains unsaturation, show limited efficacy in inhibiting [35S]TBPS binding. An inhibition 35 of [ S]TBPS binding indirectly indicates an agonistic action on the GABAA receptor. With 5β, all of the 3β-substituted derivatives of pregnanolone inhibit TBPS via a single class of binding sites. In addition, all of the 3-substituted 5β- sterols tested are full inhibitors of [35S] TBPS binding (Hogenkamp et al., 1997). Electrophysiological measurements using α1β2γ2L receptors expressed in oocytes showed that 3β-methyl- and 3β-(azidomethyl)-3α-hydroxy-5α-pregnan-20-one are potent full efficacy modulators and that 3α-hydroxy-3β-(trifluoromethyl)-5α- pregnan-20-one is a low-efficacy modulator (Hogenkamp et al., 1997). These results indicate that protection of the OH-group at the 3α position by modification of the 3β-position in 3α5αP and 3α5βP maintains activity at the neuroactive steroid site on the GABAA receptor. In animal studies, (CCD 1042) is an orally active anticonvulsant steroid, while the naturally occurring progesterone metabolites 3α5αP and 3α5βP are inactive when administered orally, suggesting that 3β-substitution slows metabolism of the 3-hydroxyl, resulting in orally bioavailable steroid modulators of the GABAA receptor.

Moreover, another substitution at 3β position; 3β-(p-acetylphenylethynyl)-3α- hydroxy-5α-pregnan-20-one (Co 152791) was more potent than 3α5αP both in [35S]TBPS binding in human recombinant receptor and Xenopus oocyte expressing

38 GABAA receptor. Ganaxolone (CCD 1042), another 3β-methyl-substituted analog of the endogenous neuroactive steroid 3α5αP has a high-affinity, stereoselective, as positive allosteric modulator of the GABAA receptor complex and exhibits potent anticonvulsant activity across a range of animal procedures. The profile of anticonvulsant activity obtained for ganaxolone supports clinical evaluation of this drug as an antiepileptic therapy with potential utility in the treatment of generalized absence seizures as well as simple and complex partial seizures (Carter et al., 1997).

Screening for the selective antagonists of neuroactive steroid potentiation of GABA responses is going on in this lab and many other labs. Some success has been achieved in this regard. An steroid analogue 3α,5α-17-phenylandrost-16-en- 3-ol(17PA) blocked the 3α5αP-enhanced GABA response and direct GABA- mimetic effect of 3α5αP (Mennerick et al., 2004). However, 17PA had no or little effect on 3α5βP steroid. The main drawback of this substance is that the results cannot be reproduced because of difficulty to dissolve and there is no effect up to 5 µM concentration. Furthermore, this substance demands further evaluation in the animal behavior model.

6.2.3 Mechanism of action of 3β-hydroxy steroids How 3β-hydroxy steroids act on GABAA receptor is not fully understood. Although 3β-hydroxysteroids reduced the potentiation induced by 3α- hydroxysteroids, 3β-hydroxysteroids act noncompetitively with respect to potentiating steroids and inhibited the largest degrees of potentiation most effectively. The profile to block activation dependant inhibition was similar to that exhibited by sulfated steroids, known antagonist of GABAA receptors (Wang et al., 2002). These antagonist neuroactive steroids are also direct, noncompetitive GABAA receptor antagonists in recombinant receptors expressed in Xenopus oocytes but this was not seen when chloride uptake was studied using synaptosomes from rat cortical tissue (Lundgren et al 2003). 3β-hydroxysteroids also inhibit the potentiating effect of barbiturates and flunitrazepam in the oocyte model. Therefore, 3β-hydroxypregnane steroids are not direct antagonists of potentiating steroids but rather are noncompetitive, likely state-dependent, blockers of GABAA receptors (Wang et al., 2002).

3β-hydroxysteroids and pregnenolone sulfate-like GABAA receptor antagonists block GABAA receptors more effectively under conditions with open channels suggesting that the antagonism of GABA responses may represent ligand- dependent or state-dependent block. This direct non-competitive effect of 3β- hydroxysteroids and pregnenolone sulfate on the GABA-response was sufficient to account for the apparent antagonism of agonist steroids. However, if the effect

39 of 3β-hydroxysteroids and pregnenolone sulfate share the common binding property is still unknown. Because of the potential importance of antagonist steroids as experimental and clinical use, it is essential to characterize the functional and binding properties of 3β-hydroxysteroids and pregnenolone sulfate.

7. Recombinant GABA receptor: Advantages and disadvantages with studies on recombinant receptors expressed in oocyte over cell lines Although other expression systems like cell line have been developed, certain experiments are only feasible in oocytes. The expression of foreign proteins in Xenopus oocytes has many advantages for electrophysiological measurements. Numerous combinations of GABA as well as other receptor subunits can be expressed in the Xenopus oocyte. It is therefore possible to obtain results from several subunit combinations and including mutated receptor subunits. The size of ooctyes is large (~1 mm) which easily accommodates manipulations like mRNA injections and electrode penetration is certainly one aspect. However, the fact that it is possible to obtain oocyte attached patch clamp recordings (Methfessel et al., 1986) and, in particular, recordings from macropatches (Stuhmer et al., 1987) is among the main advantages. The low noise recordings from a large number of channels would be difficult to obtain from other systems, and measurements, like gating current fluctuations (Conti and Stuhmer, 1989) are presently limited to the oocyte expression system.

There are some drawbacks of the oocyte expression system. There is uncertainty in homogenous results in the use of different batches of oocytes and different preparations of mRNA. These factors contribute to the diversity of results, especially when one tries to quantify them. The oocytes from different donors may differ significantly in the levels of internal second messengers and in resting membrane properties; both factors may affect the properties of the exogenous channels (Dascal, 1987). This is an inevitable disadvantage of the method, and one has to aware of it when trying to draw any generalized conclusions. The only way to avoid mistake that may be introduced by such variability is to compare as many batches of oocytes and RNA as possible. Another disadvantage is that the size to the oocyte hinders rapid applications of ligands to the whole oocyte and makes it difficult to use for the kinetics of the receptor function.

The mammalian cell expression systems also offer many advantages over other expression systems such as Xenopus oocytes. For instance, a large number of the transfected mammalian cells can be generated and passaged by standard culture techniques. Stable transfection is feasible in mammalian cells as opposed to Xenopus oocytes that can only be transiently transfected. Stable transfection in cell

40 clones shows fewer variations between experiments and batches of cells. However, to study the recombinant combinations of different GABAA receptor in a shorter time frame as it was done in these current pharmacological studies, Xenopus oocytes is the better choice.

8. Uses of rodent models for the testing of drugs for preclinical evaluation To date, the behavioral and neurological studies on effects of active GABAA receptor modulators, for example neuroactive steroids, are performed in rat models for human disorders (Johansson et al., 2002; Lofgren et al., 2006; Miczek et al., 2003; Smith et al., 1998; Turkmen et al., 2004). It is a general concept that the response of neuroactive steroids in human would be similar as in rat. There are also arguments against this concept. As the GABAA receptor modulation is dependant on the subunit combination, the pharmacology and steroid modulation of the mammalian GABAA receptor might vary between two species. It is therefore essential to investigate differences in receptor pharmacology and steroid modulation between the human and rat GABAA receptor. There are few evidences showing the difference of receptor pharmacology between the human and rat species. In a previous study by Nguyen and co-worker 1995 showed that modulation of GABAA receptor binding site in human brain by neuroactive steroids and the effects compared to those in rat brain. Unlike rat, human brain binding of [3H]flunitrazepam to the benzodiazepine site was not enhanced by alphaxalone (at any concentration), but was unaffected in many regions and inhibited in others. Binding of [3H]muscimol to high and low affinity GABA sites were enhanced by both steroids in all tested regions of rat brain (Nguyen et al., 1995). Another study showed that the tonic conductance in rat cerebellar granule cells (CGS), in contrast to that in mouse CGSs, is relatively insensitive to 3α5αTHDOC (Hamann et al., 2002). Species differences are consistent with the existence of GABAA receptor subtypes that differ in pharmacology. On the other hand, the differences in GABA receptor properties between human and rat brain, not due to the major differences in the gene sequence (Tyndale et al., 1994), but apparently in the ratios of the subunit expressed in a given brain area as indicated by binding heterogeneity (Nguyen et al., 1995). This piece of evidence suggests caution in use of rodent models for the testing of drugs such as antiepileptics or anxiolytics for use in human. Careful consideration of the similarities and differences between species may allow use of laboratory animals for certain types of preclinical research questions.

41 Aims: The overall goal of the present work was to study the effects of neuroactive metabolites of sex and stress hormones in recombinant GABAA receptor.

The specific aims of different papers were:

1. To investigate the pharmacological properties of α1-, α4- and α5-subunit containing GABAA receptors.

2. To study if the binding property of pregnenolone sulfate (PS) is different from that of Zn2+.

3. To study if the pharmacological property of 3β-hydroxysteroids is different from that of pregnenolone sulfate.

4. To study if the neuroactive steroids activity is different between the recombinant human and rat α1β2γ2L GABAA receptors.

5. To investigate the activity of neuroactive steroids between 2L (long form) and 2S (short from) variant of γ-subunit of human GABAA receptor.

42 Method Chemicals The composition of the standard ND96 solution was (in mM): 96 NaCl, 1 KCl, 1 MgCl2, 2 CaCl2 and 5 HEPES at pH 7.4. Pentobarbital was dissolved in 0.1 M NaOH as a stock solution of 25 mM, and subsequently diluted with ND96. Zn2+ was dissolved in water. The 3α-hydroxypregnane steroids used in this project were 3α-5α- androstane carbonitrile (3α5αACN); 3α-OH-5α-pregnan-20-one (allopregnanolone, 3α5αP); 3α-OH-5β-pregnan-20-one (pregnanolone, 3α5βP), 5α-pregnan-3α-ol-11,20-dione (Alphaxalone); 4-pregnan-3α-ol-20-one, 5α- androstane-3α,17β-diol (3α5α-ADL); 5α-pregnan-3α,21-diol-20-one (3α5αTHDOC); 5β-pregnan-3α,21-diol-20-one (3α5βTHDOC); 5α-pregnane- 3α,20β-diol and 5β-pregnane-3α,20α-diol. The 3β-hydroxypregnane steroids were 3β-OH-5α-pregnan-20-one (UC1010), 5α- pregnan-3β, 20β-diol (UC1011), 5β-pregnan-3β, 20α-diol (UC1013), 3β-OH-5β- pregnan-20-one (UC1014), 5β-pregnan-3β, 21-diol-20-one (UC1015), 5α-pregnan- 3β, 20α-diol (UC1019) and 5β-pregnan-3β, 20β-diol (UC1020). Steroids were dissolved in dimethyl sulfoxide (DMSO) except pregnenolone sulfate which was dissolved in the distilled water. All chemicals were then diluted in external solution for experiments. We tested the effect of DMSO in GABAA receptor expressed in Xenopus oocytes and did not find any significant response of DMSO up to 0.2% in the α1β2γ2L receptor. However, the concentration of DMSO in experimental solutions was ≤ 0.1%. cDNA cloning The cDNAs encoding the rat GABAA-receptor subunits α1, β2 and γ2 were provided by A. Tobin, University of California, Los Angels, CA, USA (α1), P. Malherbe, Hoffman-La Roche, Switzerland (β2), C. Fraser, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD, USA (γ2L). The cDNAs encoding for rat α4, α5 and of all human subunits were prepared in this lab (for details see individual articles). These plasmids have been used successfully for in vitro transcription where mRNAs were injected into Xenopus oocytes, and produced receptors were examined with electrophysiology.

In vitro transcription and expression in the Xenopus oocyte Sexually mature female Xenopus laevis were kept in optimal condition by authorized animal keepers in a local animal house and fed with standard frog food. Experimental protocols were approved by the Animal Experimentation Ethical Committee in Umeå. Stage V-VI oocytes were harvested under 0.1% tricaine (3- aminobenzoic acid ethyl ester) anesthesia. mRNA (20-40 ng total RNA) was injected 24 hours following defolliculation. Initial studies on oocytes injected with

43

Figure 10. Female Xenopus laevis frog and its oocyte

α5, β2 and γ2L mRNA molar ratio of 1:1:1 revealed a variable level of GABA EC50s and Imaxs after repeated tests. Comparatively, stable and consistent EC50s and Imaxs were observed in 2:1:1 and 3:1:1 ratios in three repeated (5~7 cells in each) experiments. On the contrary, α1, β2, and γ2L subunit mRNAs injected in equimolar ratio (1:1:1) showed a stable GABA EC50s and average Imaxs after repeated tests. Therefore, the experiments in these studies were performed at molar ratio of 2:1:1 in the α5β2γ2L and molar ratio of 1:1:1 in other rat wild-types and mutant receptors as well as human receptor.

A Nanoject II auto nanoliter injector (Drummond Sci. co., PA, USA) was used for mRNA microinjection. Oocytes were incubated up to 5 days at 18 °C in the standard ND96 at pH 7.4, supplemented with pyruvate (5 mM), (100 U/ml), streptomycin (100 µg/ml) and gentamycin (50 µg/ml).

Oocyte electrophysiology

Figure 11. Oocyte electrophysiology with voltage clamp technique

Two-electrode voltage-clamp whole-cell recordings were performed with a Warner OC725 amplifier (Warner instrument corporation, Hamden, CT, USA) 2-3 days following RNA injection. The extracellular recording solution was ND96 medium with no supplements. Intracellular recording pipettes were filled with 3 M KCl and

44 had open tip resistances of ~ 1 MΩ. Drugs were bath applied from a common tip via a gravity-driven multibarrel drug-delivery system-ValveLink 16 pinch valve perfusion system which was controlled by a Valvelink 16 controller. Cells were clamped at -70 mV for all experiments, and the steady-state current at the end of 20s drug applications was measured for quantification of current amplitudes. At the steady-state, the open and close state of Cl- channels reached equilibrium. We measure the response at the end part of drug application because of relatively slow rate of drug application. In this stage the response of lower concentrations gives a relatively horizontal and linear response and the peak and steady response are almost identical in height. However, peak response is relatively higher than steady- state response at higher concentration. The interval between the drug applications was 90 s.

Data analysis Data acquisition and analysis were performed with pClamp software (Axon Instruments, San Francisco, CA, USA). Data plotting and curve fitting were done with Sigma Plot software (SPSS, Chicago, IL, USA). Modulation of GABA- activated current was calculated as I = (IM - IN)/IN, where IN (Normalizing current) and IM (measured current) are the amplitudes of the chloride current in the absence and presence of the test drugs, respectively. Fitting of the dose-response relationships was performed using the Hill equation.

Statistical analysis Unless otherwise specified, statistical differences were determined using a two- tailed Student's t test. Analysis of variance (ANOVA) was used to analyze the effect of 3α and 3β-steroids between the human and rat α1β2γ2 receptors. With significant results (p< 0.05), Least Significant Difference (LSD) post-hoc test was used. Repeated measures analysis of variance (ANOVA) was also used to compare the efficacies of neuroactive steroids within each receptor (p< 0.05). Dose- response curve were fitted to average curve values using the Hill equation as n n n follows: I = Imax × {C /(EC50 + C )}, where C is the concentration of drug, Imax is the maximum current amplitude, EC50 is the concentration of drug that produces

50% of Imax, and n is the Hill coefficient.

All the values in the text and figures as mean ± S.E.M. Significance level used was p<0.05. The SPSS statistical package (SPSS, Chicago, IL, USA) version 12 was used for data analysis. For plotting of curves SigmaPlot 8.0 was used.

45 Results

GABA site antagonism and pentobarbital inhibition by bicuculline is not dependant on α-subunit (Paper I) The effect of bicuculline methiodide (BMI), an antagonist in the GABAA receptor binding site was compared in recombinant GABAA receptors containing α1-, α4- and α5-subunits. We found that the dose-inhibition curve of bicuculline to inhibit the sub-maximal (30 µM) GABA response was identical with the α1β2γ2L, α4β2γ2L and α5β2γ2L receptors (Paper I, figure 2a). Inhibition of the pentobarbital-activated direct currents by bicuculline was also observed in above mentioned receptor combinations. Likewise, the potencies of bicuculline to inhibit sub-maximal (1 mM) pentobarbital response were equivalent with the α1β2γ2L, α4β2γ2L and α5β2γ2L receptors (Paper I, figure 2b). The efficacies of bicuculline in terms of maximal inhibition of the 1 mM pentobarbital response were also identical (about 65% from control) in these receptors. Moreover, the presence of a fixed concentration (10, 30 or 100 µM) of bicuculline shifted the GABA-response curve to the right without decreasing the maximum response at the α1-, α4- and α5-subunit containing receptors. This type of inhibition is called competitive antagonism. On the other hand, the bicuculline inhibited pentobarbital activated current in a non-competitive manner; depression of maximum response with no significant change in EC50 (Paper I, figure 3).

Pregnenolone sulfate response in not dependant on γ-subunit (Paper II) Pregnenolone sulfate, like 3β-OH steroids, is a potent inhibitor of GABA- mediated and 3α5αTHDOC-mediated response. The dose-inhibition curves of PS to inhibit GABA and to inhibit pentobarbital response at the α1β2γ2L and α1β2 receptor combination were observed. The efficacy and potency of pregnenolone sulfate to block equivalent concentration of GABA or pentobarbital was identical at the ternary α1β2γ2L and binary α1β2 receptor (Paper II, Figure 3a and 3b). This result indicated that the γ2-subunit did not affect the potency of PS to inhibit GABAA receptor-mediated currents.

Pregnenolone sulfate and Zn2+ do not share the same binding site (Paper II) The inhibitory effect of pregnenolone sulfate was characterized in two mutant receptors- α1V256Sβ2γ2L and α1β2A252Sγ2L. In these two mutants, valine and residues at 2’position closest to the cytoplasmic end of the M2 helix were replaced by serine at α1 and β2 subunit, respectively. In both of these mutant receptors the inhibitory effect of pregnenolone sulfate to inhibit GABA was

46 significantly reduced (Paper II, figure 4e and 4f). Unlike PS, Zn2+ inhibition was not reduced at the mutant α1V256Sβ2γ2L and α1β2A252Sγ2L receptors and their degree of inhibition was similar to that with the wild-type α1β2γ2L receptor (Paper II, figure 4d). This indicates that pregnenolone sulfate does not share the same binding site of Zn2+ and that PS effects is influenced by the amino acid changes at the mutation sites.

Functional differences between 3β-hydroxysteroids and pregnenolone sulphate (Paper III) Homologous mutation of the residue at 2’position closest to the cytoplasmic end of the M2 helix to serine on both α1 and β2 subunit, α1V256S and β2A252S, reduced the slow desensitization components of GABA-activated currents at saturating doses. Compared to the wild type receptor, the potency of GABA increased significantly at the α1V256Sβ2γ2L receptor (P < 0.05), whereas it decreased moderately at the α1β2A252Sγ2L receptor (paper III, Fig. 2). We found that PS, 5α-pregnan-3β, 20(S)-diol (UC1019) and β-pregnan-3β, 20(R)-diol (UC1020) were potent antagonists at the wild type receptor. The inhibitory effect of PS was significantly reduced at both mutant α1V256Sβ2γ2L and α1β2A252Sγ2L receptors (P < 0.05), whereas the inhibitory effects of UC1019 and UC1020 were reduced only at the mutant α1V256Sβ2γ2L receptor (paper III, Fig. 3) but not at the α1β2A252Sγ2L receptors. PS promoted slow desensitization with prolonged GABA application in a dose-dependent manner at the wild type receptor, but not at the mutant receptors. On the contrary, UC1019 and UC1020 (≤ 20 µM) did not promote desensitization with both wild type and mutants receptors.

The neuroactive steroids activity is different between the recombinant human and rat α1β2γ2L GABAA receptors (Paper IV) The effect of agonist (3α-OH) or antagonist (3β-OH) neuroactive steroid series was also investigated at human and rat α1β2γ2L to find if they show any difference between the species. Both the potencies and efficacies of GABA were significantly higher at the human α1β2γ2L receptor. Enhancement of GABA EC15 response by 3α-OH-5β-pregnan-20-one (3α5βP), 5α-androstane-3α,17β-diol (3α5αADL) and 5α-pregnane-3α,20β-diol (3α5α-diol) was significantly higher at the rat receptor compare to the human receptor (paper IV, figure 3, Table 2). 3α5αP at the human and ∆4-pregnen-3α-ol-20-one at the rat receptor are more potent among agonist steroids. Additionally, diol-containing steroids showed lower degree of potentiation in both receptors. The antagonistic effects of 5α- pregnan-3β,20β-diol (UC1011) and 5α-pregnan-3β, 20(S)-diol (UC1019) at the

47 human receptor were significantly higher to inhibit the 3α5αTHDOC + GABA response compared to the rat receptor and pregnenolone sulfate in the rat receptor compared to the human receptor (paper IV, Fig. 4). Inhibition of 30 µM GABA response by 3β-OH-5α-pregnan-20-one (UC1010) and 5β-pregnan-3β, 21-diol-20- one (UC1015) was significantly higher at the human receptor compared to that in the rat and 5β-pregnan-3β, 20(R)-diol (UC1020) at the rat receptor compared to the human receptor (P <0.05).

Neuroactive steroids activity differs between 2L (long form) and 2S (short from) variant of γ-subunit of human GABAA receptor (Paper IV) Potentiation by of 3α5αTHDOC (0.1~3 µM) and 3α5βP (0.1~1 µM) was significantly higher with the γ2S isoform compared to the γ2L isoform (Paper IV, Figure 5). Moreover, inhibition of 30 µM GABA by UC1010 was significantly higher with the receptor containing γ2L and UC1020 in the receptor containing γ2S isoform, respectively. To inhibit 3α5αTHDOC + GABA response, effects of UC1010 and UC1019 were significantly higher with the γ2L isoform (P <0.05 Paper IV, Figure 5). However, GABA dose-response curves were identical for human GABAA receptors with γ2S and γ2L subunits except for an increased maximum GABA-evoked current at the γ2L subunits. Conversely, the direct effect of pentobarbital was significantly higher with the γ2S isoform.

Discussion

GABA and pentobarbital inhibition by bicuculline at α1-, α4-, α5- subunit containing GABAA receptor Our results showed that neither GABA site antagonism nor the pentobarbital inhibition by bicuculline is dependant of α-subunit. GABA-site inhibition by bicuculline at the α1-containg receptors is called phasic inhibition and inhibition at the α4- and α5-contaning extrasynaptic receptors is called tonic inhibition. It was reported earlier that the tonic inhibition observed at the α4- and α5-containing GABAA receptors was known to be more resistant to the GABAA receptor antagonists compared to the phasic inhibition in the α1-containing receptors (Farrant and Nusser, 2005; Stell and Mody, 2002). We did not find any functional difference of bicuculline between recombinant rat GABAA receptors mediating phasic and tonic inhibition since high concentrations of bicuculline in the micromolar range were tested in the present study. At submicromolar concentrations, several competitive and non-competitive GABAA antagonists (gabazine, picrotoxin and bicuculline) reduced phasic currents, but had no effect

48 on tonic currents. Moreover, the antagonists blocked both phasic and tonic currents at high concentrations (Bai et al., 2001; Semyanov et al., 2003; Stell and Mody, 2002; Yeung et al., 2003). However, low submicromolar concentrations were not tested in the present study. In our study, pentobarbital was a direct activator of α1-, α4- and α5-containing receptor and bicuculline inhibited the pentobarbital-activated currents in a non- competitive manner. However, another report demonstrated a lack of direct activation of the recombinant human α4β1γ2 receptor by pentobarbital in the oocyte expressing system (Wafford et al., 1996). On the other hand, in agreement with our findings, direct activation of α4-containing receptors by pentobarbital in the present study is with a previous study with direct activation of human α4β2γ2L receptors expressed in Xenopus oocyte by pentobarbital (Whittemore et al., 1996). Our studies also showed that the effect of GABA is dependent on γ-subunit. The potencies of GABA at the α1β2, α4β2 and α5β2 binary receptors were significantly higher compared to the ternary α1β2γ2L, α4β2γ2L and α5β2γ2L receptors. The efficacy of GABA currents evoked at the binary α1β2 and α4β2 receptors were equivalent to those evoked at the ternary α1β2γ2L and α4β2γ2L receptors. However, the α5β2 receptor possessed significantly lower GABA efficacy compared to the α5β2γ2L receptor. This indicates that for proper sorting, assembly and expression three subunits are essential for α5-containing receptor (Burgard et al., 1996).

Pregnenolone sulfate response is not dependant on γ-subunit The effect of pregnenolone sulfate at the binary α1β2 in this study is not significantly different from ternary α1β2γ2L receptor. Therefore, the inhibition by pregnenolone sulfate was not primarily dependent on the γ2-subunit. The presence of serine at the 2’ residue N-terminal to the beginning of M2 helix at both mutant α1 and β2-subunit significantly reduced PS inhibition on the GABAA receptor-mediated current. Amino acid residues underlying the inhibition of Cl- channel by PS may locate at channel lining segments linked to V256 at the α1 and A252 at the β2 subunits. Zn2+ is also a potent inhibitor of GABA binding site but its efficacy dependant on the absence of γ subunit. Mutations of amino acid residues in the above positions at the α1 and β2 subunits did not abolish the inhibitory effect of Zn2+ indicating that Zn2+ dose not share the same binding with pregnenolone sulfate. Moreover, an earlier study showed that Zn2+ has four binding site; one is located within the ion channel, other two are on the external amino (N)-terminal face of the receptor at the interfaces between alpha (E137 & H141) and beta (E182) subunits (Hosie et al., 2003). A recent study showed that four residues in the first transmembrane domain are required for the majority of the sensitivity to PS, but a charged extracellular residue at the end of the M2 helix

49 also plays a role (Wardell et al., 2006). These residues are also different from the residues responsible for Zn2+ binding.

3β-hydroxysteroids and pregnenolone sulfate inhibit recombinant rat GABAA receptor through different mechanisms Pharmacological analysis of antagonist steroids in the recombinant GABAA receptors has provided insight about functional differences between 3β- hydroxysteroids and pregnenolone sulfate. Our results suggest that the inhibition by PS, but not in the same degree by 3β-hydroxysteroids, on the GABAA receptor was dependent on the receptor desensitization. We further showed that a point mutation at M2 helix of the β2-subunit (β2A252S) can reduce the inhibitory effect of PS on the GABAA receptor without affecting the inhibitory properties of 3β- hydroxysteroids. Given that both PS and 3β-hydroxysteroids inhibit GABAA receptor-mediated currents in an activation-dependent manner, it is possible that PS inhibits GABAA receptor function by promoting receptor desensitization, whereas the inhibitory properties of 3β-hydroxysteroids are more based on other channel properties of the GABAA receptor.

The neuroactive steroids activity between the human and rat α1β2γ2L recombinant GABAA receptors Some differential response agonist and antagonist neuroactive steroids have been observed between the human and rat α1β2γ2L receptor. Some steroids showed more effect in the human receptor than rat receptor or vice -versa. Although very few reports are available regarding the comparative study between the species, one report corresponds to our finding that the neuroactive steroids effects vary between human and rat brain (Nguyen et al., 1995). Modulation GABAA receptor binding site by 3α5αTHDOC and alphaxalone varied between the human and rat brain in that study. The effects of neuroactive steroids in human disorder have been evaluated in the animal behavioral model (Johansson et al., 2002; Lofgren et al., 2006; Turkmen et al., 2004) and it is generally believed that the effects of neuroactive steroids on the GABAA receptor are also similar in human. Our results along with the above report suggest that the effect of neuroactive steroids in the rat model might be different from that in human. This heterogeneity between species provides the information that testing of potential should be made in human receptors rather than rat receptors. That may give insights to how to interpret the results from animal models and shows that result in animal studies should take into account differences between species. Our findings also suggest that further studies are clearly needed to find out any similarity and difference of other pharmacological agents between human and rat GABAA receptor.

50 Differential response of neuroactive steroids activity between 2L (long form) and 2S (short from) variant of γ-subunit of human GABAA receptor Our results show some difference of neuroactive steroid modulation between 2L (long form) and 2S (short from) of γ-subunit. This may be due to the part missing in the γ2S form of γ-subunit. γ2L differ from γ2S by having an additional 24 nucleotide exons which encodes an octapeptide (Whiting et al., 1990). The octapeptide includes a consensus phosphorylation site for PKC, with a serine in position 343. Inclusion or exclusion of this octapeptide and its phosphorylation site may alter the phosphorylation of resulting receptor and lead to altered receptor function (Kellenberger et al., 1992; Lin et al., 1996; Moss et al., 1992). Therefore, it was predicted that there would be difference in the neuroactive steroids effect between these two variant of γ-subunit. It was also reported that prolong application of some therapeutic agents can change the γ2L/S mRNA ratios. Prolong application of pentobarbital reduces the ratio. However, it was evident that 3α5αP has a little effect on the γ2L/S ratio (Tyndale et al., 1997).

51 Conclusions

GABA site antagonism and pentobarbital inhibition by bicuculline is not dependant on α-subunit. GABA potency (not efficacy) is dependant on γ-subunit. Pregnenolone sulfate (PS) response is not dependant on γ subunit and PS and 2+ Zn acts on GABAA receptor through two separate mechanisms.

3β-hydroxysteroids and pregnenolone sulfate inhibit recombinant rat GABAA receptor through different mechanisms. The neuroactive steroids activity is different between the recombinant human and rat α1β2γ2L GABAA receptors. Neuroactive steroids activity differs between 2L (long form) and 2S (short from) variant of γ-subunit of human GABAA receptor.

52 Acknowledgement

I would like to express my sincere gratitude to all the people who have supported me throughout this work. In particular, I would like to thank:

Associate professor Ming-De Wang, my supervisor, who with patience, scientific skill and generosity guided me into the world of neuroscience. Thank you very much for giving me time in spite of your intense clinical works. Without your help I could not come to this point.

Professor Torbjörn Bäckström, my co-supervisor for accepting me as a PhD student in his group. With a never-ending enthusiasm, encouragement and pedagogical skill you have helped me a lot to acquire scientific works. Thank you very much for your kindness and warm heart.

Associate professor Inga-Maj Johansson, thank you for your generous ideas, valuable comments and discussion and proof-reading of my manuscripts.

Monica Isaksson, Every time I needed the mRNA, you made that for me. It was so kind of you to show me all the technical steps of cloning and producing mRNAs. Professor Ann Lalos, head of the Department of Obstetrics and Gynecology for her friendly support. Dr. Gianna Ragagnin, thank you for the fruitful discussions about the chemical structures of steroids and friendship and of course for ‘tiramisu’. Associate professor Per Lundgren, thank you for your cooperation in the lab and help me to find references. Dr. David Haage, thank your for your fruitful discussion about ion channels. Elizabeth Zingmark and Agneta Andersson, for all the help in the laboratory assistance. Dr. Magdalena Taube, thank you very much for the friendship and keeping our lab environment so live. Jessica Stromberg and Charlotte Lindblad, for friendship and assistance in laboratory and personal issues from the beginning of my study.

Magnus Löfgren, we always shared our feeling each other and had common views in many worldly matters. Tobias Näslund, for friendship and assistance during your stay at the UNC. Birgit Ericson and Carina Jonsson, for your kind administrative support.

53 My friends and present and former co-workers at UNC, Di Zhu, Sahruh Turkmen, Vita Birzniece, Sigrid Nyberg, Lotta Andreen, Erika Timby, Anna-Carin Wihlbäck and all the fantastic people at the department of obstetrics and gynecology. Marianne Borgström and Lena Gustavsson, for taking good care of the frogs and for assistance. Finally to my wife Tanzina Chowdhury and kid Yazdan Rahman (Evan), for your endless support throughout my whole life. This work would have not been finished without your love and understanding.

This study was done at the Umeå Research Centre (UNC) under the Department of Clinical Science, Obstetrics and Gynecology, Umeå University and supported by the Swedish Research Council-medicine (project No. 4X-11198; 73p-15450), Insamlingsstiftelsen för medicinsk forskning vid Umeå Universitet, Svenska läkaresällskapet, Socialstyrelsens forskningsfond, Umeå Kvinnoklinikens forskningsfond and the EU regional fund’s objective 1 program.

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